Growth hormone releasing hormone (GHRH) and the

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Growth Hormone Releasing Hormone

(GHRH) and the GHRH Receptor

Karen Lin-Su, M.D. and Michael P. Wajnrajch, M.D.

Division of Pediatric Endocrinology

Weill Medical College of Cornell University, New York, NY

Correspondence to:

Michael P. Wajnrajch, M.D.

Weill Medical College of Cornell University

1300 York Avenue

New York, NY 10021

Tel. 212-746-3456

Fax. 212-746-0300

Email: [email protected]

GHRH and GHRH Receptors

Introduction

Linear growth in humans and other vertebrate organisms is highly dependent on growth

hormone (GH), a polypeptide hormone produced by the somatotropes of the anterior

pituitary gland. While the primary action of GH is to stimulate skeletal and visceral

growth, it has important metabolic actions as well. It is an anabolic hormone that

increases cellular amino acid incorporation into protein. It is also a lipolytic hormone,

activating lipase and thereby mobilizing fat from adipose tissue. Lastly, GH plays an

important role in carbohydrate metabolism, antagonizing the action of insulin at the post-

receptor level in skeletal muscle and adipose tissue. As a result of these actions,

deficiency of GH can result in stunted growth, delayed bone maturation, decreased lean

body mass, and hypoglycemia.

The regulation of the synthesis and secretion of GH is multifactorial, but the predominant

regulators of GH are the hypothalamic hormones, GH-releasing hormone (GHRH), GH

secretagogue (GHS), and somatostatin (SS); the former two exerting stimulatory and the

latter exerting inhibitory influences on somatotrope cells in the anterior pituitary gland.

This review will briefly describe hypothalamic control of GH as it pertains to GHRH;

discuss the structure, distribution, and function of GHRH and its receptor; and, finally,

present data about animal and human mutations that help to delineate the role of GHRH

and GHRH receptors (GHRH-R) in human disease.

Hypothalamic-Pituitary Regulation of Growth Hormone

GH is synthesized, stored, and secreted by the somatotropes of the anterior pituitary

gland. It is secreted in a pulsatile manner, with maximal secretion occurring within one

hour after the onset of sleep1. This pulsatility is believed to be an important feature of

GH in its regulation of target tissues. GH is secreted under the influence of three

GHRH and GHRH Receptors

hypothalamic hormones. The first hormone is the peptide, GHRH, which stimulates GH

synthesis and secretion via the GHRH receptor. The second hormone is the peptide,

GHS (also known as Ghrelin), which acts through the GHS receptor. The third hormone

is somatostatin (SS), which acts on the pituitary to suppress basal and stimulated GH

secretion, but is not believed to affect GH synthesis1. While the basis of the pulsatile

release of GH remains elusive, it is likely that the characteristics of the GH peaks are

determined by the combination of GHRH, GHS, and SS. Based on experiments in rats

using electrolytic lesions to eliminate SS production, it appears that SS acts by tonic

inhibition of GHRH release2. Tannenbaum and Ling3 proposed a general model of GH

secretion in which SS is secreted in a sinusoidal pattern. During the troughs of SS,

GHRH is released, thus inducing the episodic release of GH. Similarly, the peaks in SS

secretion suppress baseline GHRH release and, thus, GH release. Measurements of

GH and SS levels from direct sampling of hypophyseal-portal blood in rats support this

hypothesis, but other similar experiments in sheep failed to demonstrate this clear

relationship between SS troughs and GH pulses4,5. More recently, Roelfsema et al

studied two members of a single family with an inactivating mutation of the GHRH

receptor gene. The pattern of spontaneous GH production was noted to be preserved,

although the absolute quantity of GH secreted was quite low and the approximate

entropy significantly elevated6. This study supports the view that the timing of GH

pulses is the result of a somatostatin trough, while the amplitude of a GH pulse is the

result of a GHRH burst.

As stated previously, GH has important effects on both growth and metabolism. GH

exerts its metabolic actions directly via GH receptors on target tissues. There is also

evidence that GH has some direct effect on the growth plate7. However, much of the

linear growth produced by GH is thought to be mediated indirectly through the

generation of insulin-like growth factor-I (IGF-I), which, in turn, enhances cellular

proliferation, differentiation, and maturation of many tissues, including bone, cartilage,

and skeletal muscle. IGF-I is the predominant GH-dependent form of IGF that

GHRH and GHRH Receptors

stimulates linear growth in children, whereas IGF-II is produced in the fetus and does

not appear to be regulated by GH8. IGF-I is produced in many tissues, but the liver is

the principal source of circulating IGF-I. It appears, however, that hepatic IGF-I may not

play an essential role in growth, but rather that the relevant IGF-I is of autocrine or

paracrine origin. A liver-specific Igf-I knock-out mouse, was generated, found to have

extremely low serum Igf-I levels, but was of normal size9,10. IGF-I is transported in the

serum bound to IGF-binding proteins (IGFBPs). IGFBP-3 is one of six IGFBPs and is

the main high-affinity carrier for IGF-I. Additional regulation of GH secretion is then

achieved by feedback control from IGF-I and GH itself. Both IGF-I and GH act directly at

the pituitary to inhibit GH secretion and indirectly at the hypothalamus by suppressing

GHRH release and stimulating SS production11,12. GHS appears to regulate GH

secretion by acting directly on the hypothalamus, in addition to the pituitary (Figure 1).

GHRH

Physiology and Structure

GHRH was first isolated in 1982 from two patients who had pancreatic tumors causing

acromegaly13,14. Only later was it identified and characterized from the hypothalamus.

GHRH first appears in the human hypothalamus between 18 and 29 weeks gestation15,

which corresponds to the appearance of fetal pituitary somatrotropes. By mid-gestation,

fetal pituitary cells respond in vitro to GHRH and SS by increasing or reducing GH

secretion2. GHRH levels are high in the cord blood from term human newborns and

then decline until puberty. Girls in mid-puberty have GHRH levels five times those of

pre-pubertal girls, while pubertal boys have twice the level of GHRH than do pre-

pubertal boys. There is a further decrease in GHRH tone with advanced age16,17. The

well-recognized phenomenon of decreasing GH levels with aging, known as the

somatopause, may, in large part, be explained by this age-dependent decrease in

GHRH secretion17.

GHRH and GHRH Receptors

GHRH is a peptide hormone structurally related to the family of ‘brain-gut’ peptides,

including glucagon, glucagon-like peptide I (GLP-I), vasoactive intestinal polypeptide

(VIP), secretin, gastric inhibitory peptide (GIP), peptide with histidine as N-terminus and

isoleucine as C-terminus (PHI), and pituitary adenylyl cyclase-activating peptide

(PACAP)2,18. The GHRH peptide is C-terminally amidated in many species, including

humans, but not in rodents18.

Distribution of GHRH

GHRH-containing neurosecretory cells primarily originate from the arcuate nucleus of

the hypothalamus. Axons of these GHRH neurons project to the median eminence and

terminate on the capillaries of the pituitary portal system to stimulate GH release. Other

GHRH neurons that do not innervate the median eminence have been found in the

ventromedial nucleus, around the infundibular nucleus, in the periventricular zone, and

even the amygdala1,2. The extensive extrahypothalamic distribution of GHRH-containing

neurons in the central nervous system (CNS) suggests that, in addition to its regulation

of GH release, GHRH may play a neuromodulating role as well. Indeed, some studies

have found that GHRH promotes sleep in animals and in human male controls19-21.

Other studies demonstrated that GHRH injection stimulated food intake in rats and

sheep22,23.

Beyond its distribution in the CNS, GHRH mRNA expression has been demonstrated in

peripheral cells and tissues as well. Activity of GHRH outside the CNS was initially

detected only in carcinoid tumors and small cell carcinomas of the lung24,25. However,

GHRH was then discovered in the pancreas26 and in the epithelial mucosa of the

gastrointestinal tract27. A structurally distinct form of GHRH has been found in the

placenta, where it may serve to regulate fetal pituitary somatotropes and GH

secretion28,29. GHRH mRNA has also been detected in the ovary and testis30,31. GHRH

seems to augment follicle-stimulating hormone (FSH)-stimulated steroidogenesis in the

granulosa cells of the ovary, 32while, in the testis, its function appears to be to modulate

GHRH and GHRH Receptors

gonadotropin-stimulated steroidogenesis in Leydig cells and to amplify the action of FSH

on Sertoli cells2,18,33. Moreover, GHRH has been demonstrated in human

lymphocytes34, where it appears to serve as an immunomodulator affecting natural killer

cell activity and leukocyte migration.

GHRH Gene

The human GHRH gene was identified and characterized using GHRH mRNA from the

human pancreatic tumors in which it was originally isolated35,36. The gene has been

localized to the p12 band of chromosome 20 in the human37 and to chromosome 2 in the

mouse38. The gene includes five exons spanning about 10-18 kilobases of genomic

DNA. The placental and hypothalamic GHRH cDNA share exons 2 through 5, but their

first exons are unique. Distinct mRNAs are generated in these two tissues by the

splicing of the tissue-specific exon 1 to the common exons 2-5. These mRNAs then

encode the identical GHRH precursor proteins, suggesting that the difference in

promoters serves a solely regulatory purpose. The precursor protein for human GHRH

contains 108 amino acids, which is then processed into the 40- or 44-amino acid GHRH

peptide2 (Figure 2 ).

Mechanism of Action

GHRH is essential for the somatotrope cellular proliferation that normally occurs during

development. A decrease in GHRH production or actions is associated with

somatotrope hypoplasia. Likewise, GHRH overproduction is associated with

somatotrope hyperplasia39. GHRH is not only important for somatotrope expansion, but

for somatotrope function as well. It acts on somatotropes to increase GH biosynthesis

and secretion. Release of GHRH is episodic, in relation to SS, and accounts for the

pulsatile release of GH from the pituitary. After release from the hypothalamus, GHRH

binds to the GHRH receptor predominantly located on the pituitary somatotrope. The

occupied GHRH receptor then activates a stimulatory G- protein (Gs), composed of α-,

β-, and γ-subunits, by catalyzing the binding of the α-subunit to guanosine 5’-

GHRH and GHRH Receptors

triphosphate (GTP). The binding of GTP then stimulates adenylyl cyclase, thus

increasing intracellular cyclic 3’,5’-adenosine monophosphate (cAMP). Activation of

GHRH receptors results in the opening of a sodium channel in the somatotrope, leading

to its depolarization. The resultant change in the intracellular voltage in turn opens a

voltage-gated calcium channel, allowing the influx of calcium, which directly causes the

release of pre-made GH, stored in secretory granules1. The elevation in cAMP also

stimulates protein kinase A which phosphorylates and activates the transcription factor

CREB (cAMP response element binding protein), which then stimulates de novo GH

production via transcription of the GH1 gene as well as increasing transcription of the

GHRH receptor gene as part of a short positive feedback loop (Figure 2). 1,2,18

GH Secretatogues and Ghrelin

In addition to the primary hypothalamic regulatory hormones, GHRH and SS, a third

important regulatory hormone for GH secretion was recently identified (for more detail,

see article by ........ in this issue). Since the 1970’s, it has been known that morphine

administration resulted in GH release. By 1977, Bowers showed that enkephalins and

enkephalin analogues also caused GH secretion40 and this phenomenon was later

shown to be independent of GHRH41. This was followed by the development of a

sizable number of both peptide and non-peptide GHSs. Eventually a unique GHS

receptor was cloned from the human somatotrope and hypothalamus42, strongly

suggesting the existence of an endogenous ligand involved in controlling GH release.

That endogenous ligand was isolated in 1999 by two groups simultaneously, Kojima et

al and Tomasetto et al from the rat and mouse gastrointestinal tracts, respectively43,44.

This new protein showed great specificity for the GHS receptor and was named ghrelin

by Kojima et al and the motilin-related peptide by Tomasetto et al.

Ghrelin is located on 3q25-2645 and is composed of four exons that encode a 117-amino

acid molecule, pre-pro-ghrelin. The mature hormone, however, is a 28-amino acid

product, which is octanoylated at the third amino acid (of the mature hormone), an

GHRH and GHRH Receptors

absolute requirement for GHS receptor activation. A 27-amino acid form of ghrelin is

produced by alternative splicing, is also octanoylated at the identical position, and

appears to be equipotent to the 28-amino acid form (Figure 5). Ghrelin is primarily

produced in the oxyntic cells of the stomach and, to a lesser extent, in the arcuate,

ventro-medial, and infundibular nuclei of the hypothalamus42. Ghrelin may also be

involved in regulating food intake. In addition to its GH-secreting actions, direct

intracerebroventricular injection of ghrelin in mice has potent orexigenic action. The

orexigenicity is abrogated by a specific neuropeptide Y (NPY) Y1 receptor antagonist,

and, therefore, ghrelin appears to function through the actions of NPY, which serves to

antagonize leptin tone (which signals satiety)46. Leptin itself has been shown to

decrease GHRH receptor (and GH1) gene transcription, but to increase GHS receptor

gene transcription, yielding decreased GH levels, with decreased responsiveness to

GHRH, but increased responsiveness to GHS47. This direct transcriptional influence of

leptin on GH levels accounts for the well-recognized phenomenon of poor GH secretion

(in response to provocative stimuli) seen in obese humans.

Ghrelin regulates GH release by acting on the GHS receptor on somatotropes. The

human GHS receptor gene is located on chromosome 3p26-25 and consists of two

exons separated by over two kilobases. Two isoforms (GHS receptor 1a) and (GHS

receptor 1b) have been recognized. GHS receptor 1a is a 366-amino acid heptahelical

transmembrane protein. It is a G-protein- coupled receptor that acts via Gq11α, inositol

triphosphate (IP3), and protein kinase C48 (Figure 6). The actions of GHS are both

magnesium- and GTP- dependent. GHS receptor 1b is an alternatively spliced version

of 289 amino acids and lacks transmembrane domains six and seven as well as the

intracellular domain. The GH-secreting action of ghrelin appears, therefore, to be

entirely mediated by GHS receptor 1a. The ligand activation domain of GHS receptor

has been shown to be highly conserved from organisms as diverse as pufferfish and

humans. Assuming that proteins/domains that have been conserved for extended

periods must be physiologically important, and further assuming that the pufferfish and

GHRH and GHRH Receptors

humans diverged approximately 400 million years ago, the GHS receptor (and

presumably its ligand, ghrelin) likely plays a fundamental role in biology49.

Most studies report a synergistic effect of ghrelin with GHRH on GH release41,50-53,

although this is not universally accepted54. The presence of GHS receptors in the

hypothalamus suggests that, in addition to its direct action on somatotropes, ghrelin may

also regulate GH release by modulating GHRH and SS tone. Ghrelin mRNA is

expressed in the pituitary as early as 18 days in the rat, and its expression is highly

dependent on GHRH55. GHS serves to modulate itself by downregulating pituitary

GHSR mRNA by 50 percent; however, it does not seem to significantly alter GHRH

receptor mRNA levels39.

Although quite early in the elucidation of the GHRH/GHS relationship, there appear to

be differences in the specific roles of these parallel, but independent, pathways for GH

secretion. These include the observations that:

)

Ghrelin induces a larger release of GH than does GHRH (on a molar basis),

)

Both bolus administration and continuous GHRH infusion result in a chronic

release of GH56,

)

A bolus of ghrelin results in GH secretion, but continuous ghrelin infusion does

not; and

)

Ghrelin administration (bolus or continuous) does not cause an increase in GH1

mRNA.

It is, therefore, likely that the GHRH/GHRH receptor arm of the somatotropin pathway

serves primarily in the production of de novo GH, and secondarily in the release of

(premade) GH while Ghrelin/GHS receptors may serve primarily in the release of stored

GH, and secondarily in the production of de novo GH57,58.

GHRH and GHRH Receptors

GHRH Receptor

Structure of GHRHR

Just as GHRH belongs to a family of brain-gut peptides, the GHRH receptor belongs to

the analogous family of receptors, including secretin, VIP, glucagon, GLP-1, PACAP,

and GIP receptors. The GHRH receptor protein is 423 amino acids in length and has

seven hydrophobic transmembrane domains. Like other G-protein-coupled receptors, it

contains cysteine residues in the second and third extracellular loops that are thought to

form a disulfide bond, a cysteine in the cytoplasmic tail for palmitoylation, an amino-

terminal site for N-linked glycosylation, potential phophorylation sites in the third

cytoplasmic loop, and numerous highly conserved residues within the transmembrane

domains (Figure 4)18.

Expression of GHRH Receptors

GHRH receptors are primarily expressed in the pituitary gland and, using in situ

hybridization, have been localized to the anterior pituitary. In a murine model, Lin et al

showed that GHRH recptors are expressed in the caudal region of the anterior pituitary

in a temporal and spatial pattern corresponding to GH1 expression59. GHRH receptor

mRNA has also been detected in the placenta28,60 and the kidney61.

Levels of GHRH receptor expression in the pituitary vary with different developmental

stages. GHRH receptors are first detected on embryonic day 16.5 in the mouse, peak in

the late embryonic period, and then decline before increasing again during puberty59,62.

GHRH Receptor Gene

The human GHRH receptor gene has been mapped by polymerase chain reaction to

chromosome 7p15 by Wajnrajch et al63 and by in situ hybridization to chromosome 7p14

by Gaylinn et al64. Characterization of the genomic sequence has revealed that the

human gene consists of 13 exons and spans 15 kilobases. Introns range in length from

126 to 4708 nucleotides1. The 5’-flanking region of GHRH receptor gene contains

GHRH and GHRH Receptors

several binding sites for transcription factors, such as CREB, that modulate GHRH

expression. Furthermore, binding sites for regulatory factors that are known to bind to

the GH promoter and are responsible for GH basal activity, such as the nuclear factor,

NF-1, and the upstream regulatory factor, USF, have also been identified on the

GHRHR gene promoter1.

The mouse Ghrh receptor gene has been linked to chromosome 665, where it also

consists of 13 exons and spans more than 10 kilobases. The rat GHRH receptor gene

has a similar structure of 13-14 exons spanning about 15 kilobases66. Comparable to

the human gene, the 5’-flanking region of rat GHRH receptor gene contains a potential

binding site for the transcription factor, Pit-1 (see below), as well as for several steroid

hormone-responsive elements66.

PIT-1 and PROP-1

Within the pituitary gland, GHRH receptors are co-expressed with the pituitary-specific

transcription factor, PIT-1, in the anterior lobe. PIT-1 (official nomenclature now

POU1F1) is a member of a POU family of transcription factors, named for its members

Pit-1, Oct-1, and Unc-8667. It is involved in the developmental generation of

somatotropes and in the regulation of the genes for GH (GH1), prolactin (PRL), and the

β-chain of thyroid stimulating hormone (TSH-β). The PIT-1 gene contains three

functional domains, the first two of which are critical for high-affinity binding to these

genes. The third domain enables activation of the GH1, PRL, and TSH-β genes68. PIT-

1 plays an important role in GH regulation by activating transcription of GH1, thus

leading to increased GH1 mRNA and GH protein, and replenishment of cellular stores of

GH. In addition to its effects on the GH1, PRL, and TSH-β genes, it also stimulates

transcription of the GHRH receptor gene, leading to increased numbers of GHRH

receptors on responding somatotropes. It provides the pathway whereby GHRH leads

to increased GH synthesis, but also directly regulates synthesis of GHRH receptors

through its gene promoter69.

GHRH and GHRH Receptors

PROP-1 (Prophet of PIT-1) is a transcription factor expressed exclusively in the anterior

pituitary. PROP-1 is also essential for somatotrope (as well as thyrotrope, lactotrope,

and gonadotrope) development. It does not appear to have a direct role in gonadotrope

function, but it necessary for gonadotrope differentiation67. PROP-1 is essential during

embryonic development for the expression of PIT-170, as well as for the extinguishing of

the expression of another anterior pituitary transcription factor, HESX171.

Hormonal Regulation of GHRH Receptors

Regulation by Glucocorticoids

The regulation of the GH axis by glucocorticoid hormones is multifaceted and seemingly

contradictory. Whereas glucocorticoid deficiency results in decreased GHRH binding

and, thus, decreased GH secretion, glucocorticoid excess blunts the GH response to

GHRH and tends to suppress growth as well. Glucocorticoids are essential for pituitary

expression of GH and for somatotrope differentiation during development69. Studies in

adrenalectomized rats demonstrate a down-regulation of GHRH-binding sites in

somatotropes, which are then restored with glucocorticoid replacement72.

Glucocorticoids have been shown to stimulate GHRH receptor gene expression both in

vivo and in vitro73,74. Indeed, dexamethasone administered to normal subjects results in

a dose-dependent and time-dependent increase in GHRH receptor mRNA75.

Regulation by Gonadal Hormones

Sexual dimorphism is characteristic of GH secretion, both in humans and in rodents, and

becomes more prominent at the time of puberty. A clear increase in GHRH receptor

gene expression has been described in the rat at the onset of sexual maturation, with

female levels expressed at only 15% those of males62,76. There appears to be

significant inhibition of the GHRH receptor gene promoter by β-estradiol77. At the same

time, androgens have been shown to directly increase GHRH gene expression in the

arcuate nucleus of the hypothalamus78. Even in the neonatal period, gonadal steroids

GHRH and GHRH Receptors

begin to establish a sexually dimorphic pattern of GH secretion79 by modulating both

GHRH and SS neurons80.

Regulation by Thyroid Hormone

While there has been no thyroid hormone receptor response element found in the 5’-

flanking region of the GHRH receptor gene, there is clearly an influence of thyroid

hormone on GHRH and the GH axis. Hypothyroidism results in impaired linear growth,

which is then restored with thyroid hormone replacement. GHRH receptor gene

expression and GH secretion are both markedly reduced in the thyroid- deficient

state81,82. Thyroid hormone has been shown to enhance somatotrope sensitivity to

GHRH in vitro83, and incubation of pituitary cells with triiodothyronine (T3) leads to a

striking increases in GHRH receptor mRNA74.

Mutations in the GHRH receptor gene

Mouse Model

Identification of the little (lit) mouse has served as an important model for human GH

deficiency disorders. The little mouse expresses an autosomal recessive missense

mutation on chromosome 6 that functionally inactivates GHRH receptors, thus

preventing GHRH-induced responses. A single nucleotide transition changing an

aspartate codon to a glycine codon at amino acid position 60 was identified in the N-

terminal extracellular domain of the GHRH receptor gene38,65. The little mouse has

growth deficiency, growing to only about 60% of normal body weight69. Pituitary GH and

GH1 mRNA are markedly reduced, but still detectable in homozygous lit/lit mice84.

Levels of GHRH receptor mRNA also appear to be moderately reduced in the

pituitary38,85. There is also a decrease in the number of pituitary somatotropes, and the

few that remain do not respond to GHRH. They do, however, secrete GH when treated

with cAMP or other agents that elevate intracellular cAMP86.

Human Mutations

GHRH and GHRH Receptors

No mutations have been identified to date in the GHRH gene, while approximately 10

different deleterious mutations have been described in the human GHRH receptor

gene, all resulting in isolated GH deficiency. Wajnrajch et al87 identified a nonsense

mutation in the human GHRH receptor gene resulting in profound GH deficiency in two

members of a consanguineous family. Both subjects were found to be homozygous for

a GT transversion at position 265, resulting in an amber-type (‘TAG’) stop mutation

(Glu72Stop). The mutation was found close to the position of the lit mutation in the

mouse, with both occurring in the same highly conserved region of the extracellular

domain. The patients, as expected, failed to respond to stimulation with GHRH, but

responded well to standard GH replacement therapy. The same mutation was found in

several affected families of a large cluster of severe familial dwarfism88 and in a family

from an island just off of Sri Lanka89. Other identified mutations in the GHRH receptor

gene include the splice site mutation at intron 1, IVS1+1GA90; a 5-base pair deletion in

exon 11 (del 1140-1144)91; and four missense mutations in the transmembrane region of

the molecule (H137L, A222E, F242C, and L144H)92.

Role of GHRH and GHRH Receptors in Disease

The number of mutations now described in the GHRH receptor gene make

abnormalities of this gene one of the most common causes of isolated GH deficiency.

Likewise, there has been a suggestion that activating mutations may lead to GHRH

overexpression and play a role in pituitary adenoma formation. Transgenic mice with

chronic GHRH receptor stimulation by GHRH overexpression eventually develop

pituitary tumors93. In 40% of human somatotrope adenomas, dominant somatic

mutations of the Gsα gene (gsp oncogenes) have been found, leading to an activated

GTP-bound state and constitutive adenylyl cylase induction94,95. Up-regulation of GHRH

receptor mRNA has also been found in somatotrope tumors96.

In summary, GHRH and its receptor provide important functions in the regulation of the

GH axis and in the development and proliferation of pituitary somatotropes. Additional

GHRH and GHRH Receptors

investigation of the actions of GHRH and GHRH receptors may provide further insight

into their comprehensive roles in both normal physiology and in the pathogenesis of

human disease.

GHRH and GHRH Receptors

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Figure 1. Hypothalamic-Pituitary Regulation of Growth Hormone (GH). SS, somatostatin; GHRH,

growth hormone releasing hormone; AP, anterior pituitary; IGF-I, insulin like growth factor type I.

GH is produced and secreted by the somatotroph cells of the AP under the influence of two hypothalamic

hormones. GHRH stimulates GH and SS inhibits GH secretion. Ghrelin also stimulates GH secretion from

the AP via its own receptor. GH exerts direct metabolic effects on target tissues and exerts its growth

effects through IGF-I, which is produced primarily by the liver. Additional regulation of GH secretion is

achieved through feedback control by IGF-I and GH at the pituitary and at the hypothalamus.

HYPOTHALAMUS

GHRH SS

Target

Tissues

Liver

Direct

Indirect

Metabolic Effects

anti-insulin

lipolytic

anabolic

IGF-I

Bone, Cartilage,

Other Tissues

Growth

(-)

(+)

Stomach

Ghrelin

(-)

Figure 3. Mechanism of action of growth hormone releasing hormone (GHRH) on the pituitary

somatotrope to increase growth hormone (GH) synthesis and secretion. Binding of GHRH to its

receptor activates a stimulatory G protein, which stimulates adenylyl cyclase to increase cAMP. Increased

cAMP increases intracellular calcium, which stimulates GH release. Increased cAMP also stimulates

protein kinase A (PKA) to activate cAMP response element binding protein (CREB), which stimulates

GH1 and GHRHR gene transcription.

GHRHR

GHRH

Gsα

Adenylyl

Cyclase

ADP

cAMP

Ca2+

CREB

Promoter

GH1

Ca2+

PKA

GHRHR

3’ 5’ 2 5 4

GHRH

Gene 1b

COOH

NH2

Signal

Sequence GHRH* Carboxy-Terminal

Peptide

*Carboxy terminus amidation

Protein

Domain

Figure 2

1p 3

GHRELIN (GHS)

3’

5’

GHRELIN

1 3 2 4

* Site of octanoylation Site of alternative splicing

COOH

NH2 28 AA

Signal

Sequence

Gene

Protein

Domain

Figure 4

GHS Receptor 1a

5 ́

3’

Promotor

Gene

COOH NH2 3 2 1 4 5 6 7

Protein

Figure 5

GHRHR

GRE +

ERE -

COOH

NH2

TM 1 2 3 4 5 6 7

3’ 5’ 1 2 3 4 5 6 7 8 9 10 11 12 13

IVS1

E72X

Promoter

Figure 6

... The effects of GHRH and its analogs are mediated by pGHRH-R [2,3,9,10,15,16]. Activation of GHRH-R upon the binding of GHRH, a 44-amino acid peptide released by the hypothalamus, results in the secretion and production of GH through cyclic adenosine monophosphate (cAMP)-dependent pathways [3,14,16,17]. ...

... Alternative splicing, e.g. exon skipping, splice site selection, and intron retention, resulting in deletion, exchange, and insertion of receptor sequences is often observed among GPCRs [1,7,9,16]. Also, splice variants of many GPCRs, including GHRH-Rs, have been shown in different cancer types, however, their biological significance is poorly understood. ...

... Also, splice variants of many GPCRs, including GHRH-Rs, have been shown in different cancer types, however, their biological significance is poorly understood. Alternative splice variants of GHRH-Rs have been found under hypoxic microenvironment in solid tumors, suggesting a specific role in cellular adaptation and malignant processes [1,9]. Rekasi et al. in 2000 detected two major splice variants (SVs) of the GHRH-R in human extrapituitary cells as well as in human cancer cell lines [2,22,23]. ...

Gabor Halmos Zsuzsanna Szabó

Nikoletta Dobos Andrew V. Schally

The hypothalamic polypeptide growth hormone-releasing hormone (GHRH) stimulates the secretion of growth hormone (GH) from the pituitary through binding and activation of the pituitary type of GHRH receptor (GHRH-R), which belongs to the family of G protein-coupled receptors with seven potential membrane-spanning domains. Various splice variants of GHRH-R (SV) in human neoplasms and other extrapituitary tissues were demonstrated and their cDNA was sequenced. Among the SVs, splice variant 1 (SV1) possesses the greatest similarity to the full-length GHRH-R and remains functional by eliciting cAMP signaling and mitogenic activity upon stimulation by GHRH. In this review, we briefly discuss the activation, regulation, molecular mechanisms and signaling pathways of GHRH-Rs and their SVs in various tissues and also summarize the expression, biological activities and potential function of GHRH, its analogs and their receptors. A large body of work have extensively studied and evaluated potential clinical applications of agonists and antagonists of GHRH in diverse fields, including oncology, endocrinology, obesity, diabetes, other metabolic dysfunctions, cardiology, immune functions, mood disorders, Alzheimer’s and lung disease, ophthalmology, inflammation, wound healing and other applications. These results strongly support the potential therapeutic use of GHRH analogs in human medicine in the near future.

... and cancer cell lines derived from breast, endometrial, ovarian, prostatic, pancreatic, gastric, colorectal, lung and brain tissues, and bone sarcomas, lymphomas and renal-cell carcinomas [1,[4][5][6][7][8][9][10][11]. The hormonal activities of GHRH and its analogs are mediated by the full-length pituitary type GHRH receptor (pGHRH-R) [1,3,4]. However, previous investigations have also identified splice variants (SVs) of GHRH-Rs in human cancers and other extrapituitary tissues that can mediate the effects of GHRH and its agonistic and antagonistic analogs [1,4-11] (Figure 1). ...

... Growth hormone-releasing hormone (GHRH) is a peptide neurohormone secreted by the hypothalamus. Generally, it is known to act on the pituitary gland to stimulate the production and release of growth hormone (GH) through its binding to GHRH receptors (GHRH-R) [1][2][3][4][5][6][7]. In addition to this endocrine role, the biological actions of this 44-amino acid peptide are not limited to the pituitary-hypothalamic axis, since it was demonstrated that GHRH can function as an autocrine/paracrine growth factor in various extrapituitary tissues and several human cancers [1][2][3][4][5][6][7]. ...

Éva Juhász Zsuzsanna Szabó Andrew V. Schally

Csongor Kiss

Hematological and oncological diseases are still among the leading causes of childhood mortality. Expression of growth hormone-releasing hormone (GHRH) and its receptors (GHRH-R) has been previously demonstrated in various human tumors, but very limited findings are available about the presence and potential function of GHRH-Rs in oncological and hematological disorders of children. In this study, we aimed to investigate the expression of mRNA for GHRH and splice variant 1 (SV) of GHRH-R in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of GHRH-R protein were also studied by Western blot and ligand competition assays. Of the fifteen specimens studied, eleven pediatric samples (73%) showed the expression of mRNA for GHRH. These eleven samples also expressed mRNA for GHRH receptor SV1. GHRH-R protein was found to be expressed in two benign tumor samples and five malignant tumors examined by Western blot. The presence of specific, high affinity binding sites on GHRH-R was demonstrated in all of the seven human pediatric solid tumor samples investigated. Our results show that the expression of GHRH and SV1 of GHRH-R in hemato-oncological diseases in children can pave the way for further investigation of GHRH-Rs as potential molecular targets for diagnosis and therapy.

... IGF-I, in turn, positively feedbacks on GH secretion and modulates its receptor expression. Moreover, IGF-I affects animal reproduction, lactation, and feed intake by interacting with the somatotropic axis [32][33][34]. Both genes have been shown to play critical roles in milk production and composition in dairy cattle, with variations in these genes affecting milk output and quality [23,25,26]. ...

Elsayed E Hafez

Background The insulin-like growth factor (IGF-I) and growth hormone (GH) genes have been identifed as major regulators of milk yield and composition, and reproductive performance in cattle. Genetic variations/polymorphism in these genes have been found to infuence milk production, yield and quality. This investigation aimed to explore the association between IGF-I and GH polymorphisms and milk yield and composition, and reproductive performance in a herd consisting of 1000 Holstein–Friesian (HF) dairy cattle from El-Alamia farm. The experimental animals were 76±7.25 months in age, with an average live weight of 750±50.49 kg, and raised under the same conditions of feed‐ ing and weather. The studied animals were divided into three categories; high producers (n=280), medium producers (n=318) and low producers (n=402). Results The digestion of 249 bp for IGF-I-SnaBI using the Restriction-fragment-length-polymorphism (RFLP) tech‐ nique yielded two alleles; T (0.59) and C (0.41) and three genotypes; TT (0.52), TC (0.39) and CC (0.09) and this agrees with the results of DNA/gene sequencing technique. The sequencing analysis of the IGF-I gene revealed polymorphism in position 472 (C>T). Nucleotide sequencing of the amplifed fragment of the IGF-I gene of diferent genotypes was done and submitted to the NCBI GenBank with Accession no. MH156812.1 and MH156811.1. While the digestion of 432 bp for GH-AluI using the RFLP technique yielded two alleles; A (0.81) and G (0.19) and two genotypes; AA (0.77) and AG (0.23) and this agrees with the results of DNA/gene sequencing technique. The sequencing analysis of the GH gene revealed polymorphism in the position 1758 C>G and in turn led to changes in amino acid sequence as Ala‐ nine for (A) compared to Glycine for (G). Nucleotide sequencing of the amplifed fragment of the GH gene was done and submitted to the NCBI GenBank with Accession no. MH156810.1. The results of this study demonstrate the efects of variants of the GH-IGF-I somatotrophic axis on milk production and composition traits in commercial HF cattle. The greatest values of milk yield and reproductive performance were observed on IGF-I-SnaBI-TC and GH-AluI-AG genotypes. While the greatest % fat and % protein values were observed on IGF-I-SnaBI-CC and GH-AluI-AA genotyped individuals. Conclusion The genetic variation of the studied genes can be utilized in selecting animals with superior milk yield, composition and reproductive performance in Holstein–Friesian Dairy Cattle under subtropical conditions.

Ahmed Saleh Tarek G. M. Hassan

Dalia Kassem El-Hedainy

Amr Rashad

Background The insulin-like growth factor (IGF-I) and growth hormone (GH) genes have been identified as major regulators of milk yield and composition, and reproductive performance in cattle. Genetic variations/polymorphism in these genes have been found to influence milk production, yield and quality. This investigation aimed to explore the association between IGF-I and GH polymorphisms and milk yield and composition, and reproductive performance in a herd consisting of 1000 Holstein–Friesian (HF) dairy cattle from El-Alamia farm. The experimental animals were 76 ± 7.25 months in age, with an average live weight of 750 ± 50.49 kg, and raised under the same conditions of feeding and weather. The studied animals were divided into three categories; high producers (n = 280), medium producers (n = 318) and low producers (n = 402). Results The digestion of 249 bp for IGF-I-SnaBI using the Restriction-fragment-length-polymorphism (RFLP) technique yielded two alleles; T (0.59) and C (0.41) and three genotypes; TT (0.52), TC (0.39) and CC (0.09) and this agrees with the results of DNA/gene sequencing technique. The sequencing analysis of the IGF-I gene revealed polymorphism in position 472 (C > T). Nucleotide sequencing of the amplified fragment of the IGF-I gene of different genotypes was done and submitted to the NCBI GenBank with Accession no. MH156812.1 and MH156811.1. While the digestion of 432 bp for GH-AluI using the RFLP technique yielded two alleles; A (0.81) and G (0.19) and two genotypes; AA (0.77) and AG (0.23) and this agrees with the results of DNA/gene sequencing technique. The sequencing analysis of the GH gene revealed polymorphism in the position 1758 C > G and in turn led to changes in amino acid sequence as Alanine for (A) compared to Glycine for (G). Nucleotide sequencing of the amplified fragment of the GH gene was done and submitted to the NCBI GenBank with Accession no. MH156810.1. The results of this study demonstrate the effects of variants of the GH-IGF-I somatotrophic axis on milk production and composition traits in commercial HF cattle. The greatest values of milk yield and reproductive performance were observed on IGF-I-SnaBI-TC and GH-AluI-AG genotypes. While the greatest % fat and % protein values were observed on IGF-I-SnaBI-CC and GH-AluI-AA genotyped individuals. Conclusion The genetic variation of the studied genes can be utilized in selecting animals with superior milk yield, composition and reproductive performance in Holstein–Friesian Dairy Cattle under subtropical conditions.

... Most importantly, after one hour of transfection with plasmids containing WT or mutant GHRH, GH secretion levels stimulated by mutant GHRH were significantly decreased compared with those stimulated by WT GHRH, indicating a reduced capacity of mutant GHRH to stimulate GH secretion. As commonly understood, the activation of GHRHR by GHRH leads to the synthesis and release of GH via Gs-mediated cAMP signaling pathways (Lin-Su and Wajnrajch, 2002;Zhou et al., 2020). Our research findings indicate that the cAMP levels induced by a 1-hour stimulation of mutant GHRH were significantly reduced in comparison to those observed with WT GHRH, signifying a reduced receptor activation. ...

Ebru Ucakturk

Emirhan Nemutlu

Growth hormone-releasing hormone (GHRH) and its synthetic analogs are considered performance-enhancing substances and are therefore prohibited by the World Anti-Doping Agency (WADA). The analysis of GHRH and its analogs in urine presents significant analytical challenges due to their inherent in vivo instability, rapid renal clearance, and low urinary concentrations. The present study aimed to develop a robust nano-LC quadrupole/orbitrap mass spectrometry (nano-LC–Q/Orbitrap MS) method for both screening and confirmation analyses of GHRH and its synthetic analogs (sermorelin/CJC-1293, tesamorelin, and CJC-1295) and the primary metabolite of sermorelin in urine, in accordance with WADA requirements. The sample preparation workflow was systematically investigated. Existing solid-phase extraction (SPE) protocols were compared, and two additional commercially available SPE cartridges were evaluated. Within the SPE step, the influence of various washing and elution solvent strengths on peptide recovery was also systematically examined. The effectiveness of different cleanup solvents during the ultrafiltration step was further assessed. Based on these evaluations, a refined approach was developed, incorporating an initial ultrafiltration step followed by SPE. The proposed method was fully validated according to WADA guidelines, assessing key parameters such as selectivity, reliability, limits of detection (LOD), carryover, limits of identification (LOI), robustness, autosampler stability, and matrix effects. The validation results confirmed the method’s suitability and robustness for anti-doping testing. Achieved LODs (≤ 0.5 ng/mL) and LOIs (0.5–0.75 ng/mL) demonstrated sufficient sensitivity for effective detection and confirmation analysis of the target peptides in urine.

Fabio Bioletto

Emanuele Varaldo Valentina Gasco Silvia Grottoli

The regulation of growth hormone (GH) synthesis and secretion by somatotroph cells of the anterior pituitary is a highly complex process, mediated by a variety of neuroendocrine and peripheral influences. In particular, a key role is played by the hypothalamic peptides growth hormone-releasing hormone (GHRH) and somatostatin, which regulate the somatotroph axis with opposite actions, stimulating and inhibiting GH release, respectively. Since the discovery of GHRH about 50 years ago, many pathophysiological studies have explored the underlying intricate hormonal balance that regulates GHRH secretion and its interplay with the somatotroph axis. Various molecules and pathophysiological states have been shown to modulate the release of GH, GHRH, somatostatin and GH secretagogues. Collectively, the available evidence demonstrates how a vast number of neural and peripheral signals are conveyed and integrated to orchestrate a finely tuned response of the somatotroph axis that adapts to the body’s varying needs for growth, metabolism, and repair. The present review aims to summarize the available evidence regarding the key regulators involved in the modulation of the somatotroph axis in humans, presenting detailed molecular insights on the signaling cascades at play. The interplay between different mechanisms governing somatotroph secretion is highlighted, underscoring the nuanced interdependence that maintains homeostasis and facilitates the body’s ability to respond to internal and external stimuli.

Mervan Bayraktar Nazan Koluman

Mohammed Baqur Al-Shuhaib

This study investigates the genotype and allele frequencies of three novel SNP loci (c.4218 T>G, c.2203 T>C, and c.7966 A>C) in the GHRHR gene within the Damascus goat breed and their associations with growth traits. The genotype frequencies for c.4218 T>G were 0.65 (TT), 0.15 (TG), and 0.20 (GG), with allele frequencies of 0.73 (T) and 0.27 (G), showing a significant deviation from Hardy-Weinberg equilibrium (HWE). The c.2203 T>C locus had genotype frequencies of 0.75 (TT) and 0.25 (CC), and the c.7966 A>C locus had 0.81 (AA) and 0.19 (CC), both consistent with HWE. Association analysis revealed significant correlations between the c.4218 T>G SNP and body weight and rump width, with the TT genotype showing the highest averages for both traits. The c.2203 T>C SNP was significantly associated with body weight and chest width, with the TT genotype again showing higher averages. The c.7966 A>C SNP was associated with chest depth and body length, with the AA genotype displaying lower averages for these traits. In silico predictions using multiple computational tools indicated that the identified missense SNP (p.31Ile>Ser) is novel and likely deleterious to the GHRHR protein’s function and stability. Molecular docking demonstrated that the wild-type GHRHR binds more effectively with G protein alpha compared to the mutated form, predicting a mechanism through which this variant reduces the interactions of GHRHR with its cognate proteins. These findings provide valuable insights into the genetic factors influencing growth traits in Damascus goats and highlight the potential impact of the p.31Ile>Ser SNP on GHRHR functionality. This research underscores the importance of incorporating genetic markers in breeding programs to enhance growth traits and overall productivity

Maleeha Ahmad Robert J. Weil

Growth-hormone-producing pituitary adenomas in adults will be the focus of this review acromegaly is a disorder caused by pathologically excess levels of growth hormone (GH), nearly always secondary to a pituitary somatotroph adenoma, which account for 10–20% of all pituitary adenomas. Acromegaly is a pan-systemic disease, including but not limited to effects of excess growth hormone on the cardiovascular, respiratory, gastrointestinal, metabolic, and reproductive systems. This raises the concern for clinicians and patients alike in diagnosing the underlying disease when multiple systems are involved. Numerous organ systems may be differentially affected, and the multiplicity of signs and symptoms possibly overlap with other conditions, with the typically slow progression of the disease, it may take years from the initiation of biochemical GH excess before a diagnosis of acromegaly is made. The goal of effective treatment of acromegaly is to eliminate hypersecretion of GH and normalize the production of IGF-1 while preserving normal pituitary function. Medication, radiotherapy, and surgery, often in combination, and over time, are required to mitigate, reduce, and eliminate the morbidity and excess, premature mortality caused by GH elevation.

Sanggun Roh Gui-Ying Nie Kylie Loneragan

Chen Chen

Leptin is produced primarily in adipocytes and regulates body energy balance. A close link between leptin and pituitary hormones, including GH, has been reported. The mechanisms employed by leptin to influence somatotropes are not clear, however. Here we report a direct action of recombinant ovine leptin on primary cultured ovine somatotropes by analyzing the levels of mRNA encoding for GH or the receptors for GHRH (GHRH-R) and GH-releasing peptides (GHRP). Treatment of ovine somatotropes with leptin (10−7–10−9 m) for 1–3 d reduced the mRNA levels encoding GH and GHRH-R, but increased GHRP receptor mRNA levels in a time- and dose-dependent manner. Three-day treatment of cells with leptin decreased the GH response to GHRH stimulation, but the GH response to GHRP-2 stimulation was increased. The combined effect of GHRH and GHRP-2 on GH secretion was not altered by treatment of the cells with leptin. These results demonstrated a direct action of leptin on ovine pituitary cells, leading to a reduced sensitivi...

John S Parks Milton R. Brown

David L Hurley

Michael Paul Wajnrajch

Basic and translational research achievements over the past 2 decades have disclosed the molecular mechanisms underlying several genetic forms of hypopituitarism. Disorders that are limited to the hypothalamic, pituitary, GH axis are caused by mutations in individual components of that axis. Disorders involving GH and one or more additional pituitary hormones are caused by mutations in the homeodomain transcription factors that direct embryological development of the anterior pituitary gland. Pit-1 has a POU-specific and a POU-homeo DNA-binding domain. The phenotype produced by mutations in the PIT1 gene involves deficiencies of GH, PRL, and TSH. Pituitary glands are either small or normally sized. The PROP1 gene encodes a transcription factor with a single paired-like DNA-binding domain. Persons with inactivating mutations in PROP1 have deficiencies of LH and FSH, as well as GH, PRL, and TSH. Their pituitary glands may be small, normally sized, or extremely large and show suprasellar extension. Pituitary degeneration may produce acquired deficiency of ACTH. Expression of the HESX1 gene precedes expression of PROP1 and PIT1, and it is much more widespread. The protein has a paired-like domain, and it competes with the product of PROP1 for DNA-binding. Homozygosity for inactivating mutations of HESX1 produces a complex phenotype that resembles septo-optic dysplasia. Much more needs to be learned about the role of HESX1 mutations in other forms of hypopituitarism.

Jun Kamegai Hideki Tamura Takako Shimizu

Shinichi Oikawa

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was originally purified from the rat stomach. Ghrelin specifically releases GH following intravenous administration, and its GH-releasing activity in vivo is dependent on growth hormone-releasing hormone (GHRH). We previously reported that the expression of the GHS-R gene in the pituitary is developmentally regulated and GHRH infusion increases pituitary levels of GHS-R mRNA. Ghrelin mRNA and peptide have recently been detected in rat and human pituitaries. However, the regulation of the ghrelin gene in the pituitary is unknown. In this study, pituitary levels of ghrelin mRNA were measured with the reverse transcriptase-polymerase chain reaction in male rats at embryonic day (e)18 and postnatal days 1, 10, 30, and 75. The highest concentrations of ghrelin mRNA in the pituitary were observed at e18 and then they declined with age. The infusion of GHRH (10 μg/h, 4h) in freely-moving adult male rats resulted in a 1.9-fold increase in ghrelin mRNA levels relative to control rats (P < 0.05). These data indicated that the expression of the ghrelin gene in the pituitary is developmentally regulated and the pituitary ghrelin/GHS-R signaling system could modulate the regulation of GH secretion by GHRH. Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was originally purified from the rat stomach. Ghrelin specifically releases GH following intravenous administration, and its GH-releasing activity in vivo is dependent on growth hormone-releasing hormone (GHRH). We previously reported that the expression of the GHS-R gene in the pituitary is developmentally regulated and GHRH infusion increases pituitary levels of GHS-R mRNA. Ghrelin mRNA and peptide have recently been detected in rat and human pituitaries. However, the regulation of the ghrelin gene in the pituitary is unknown. In this study, pituitary levels of ghrelin mRNA were measured with the reverse transcriptase-polymerase chain reaction in male rats at embryonic day (e)18 and postnatal days 1, 10, 30, and 75. The highest concentrations of ghrelin mRNA in the pituitary were observed at e18 and then they declined with age. The infusion of GHRH (10 μg/h, 4h) in freely-moving adult male rats resulted in a 1.9-fold increase in ghrelin mRNA levels relative to control rats (P < 0.05). These data indicated that the expression of the ghrelin gene in the pituitary is developmentally regulated and the pituitary ghrelin/GHS-R signaling system could modulate the regulation of GH secretion by GHRH.

Andrew I. Korytko P Zeitler Leona Cuttler

The mechanisms governing age-dependent patterns of GH secretion are not well understood. Studies have shown that pituitaries of fetal and neonatal mammals are highly responsive to the stimulatory effect of GH-releasing hormone (GHRH) compared to those of mature mammals. Differential pituitary responsiveness to GHRH may, therefore, contribute to the elevated circulating GH concentrations characteristic of the perinatal period and the subsequent decline in circulating GH late in life. Age-dependent expression of GHRH-receptor (GHRH-R) would provide a cellular mechanism to direct differential somatotroph responsiveness to GHRH. Therefore, we determined the ontogeny of rat GHRH-R gene expression. We studied rats at ages that correspond to major changes in circulating GH levels: embryonic day 19.5 (of 21.5-day gestation period); postnatal days 2, 12, 30, 45, and 70; and 1 yr of age. We found that GHRH-R messenger RNA (mRNA) expression was markedly age dependent (P < 0.0003). The concentration of GHRH-R mRNA wa...

Anna Bagnato

Costanzo Moretti

Junji Ohnishi Kevin J. Catt

GH-releasing hormone (GHRH) is a potent cAMP-mediated agonist in the rat ovary, where it binds to a common vasoactive intestinal peptide/GHRH receptor and enhances the actions of FSH on granulosa cell maturation. A GHRH-like peptide has been detected by immunocytochemistry in the human ovary and by RIA in follicular fluid, suggesting local synthesis of the peptide. In rat ovarian poly(A)+ RNA, Northern blot hybridization analysis with a 32P-labeled 48-nucleotide (nt) rat GHRH oligonucleotide probe revealed the presence of one major and two minor mRNA species. The major ovarian GHRH mRNA (1750 nt) was much larger than that present in hypothalamus and placenta (750 nt), but was similar to that observed in the rat testis. Two well defined higher mol wt forms of 3.2 and 3.6 kilobases were also present and probably represent unprocessed precursors of the 1750-nt mRNA. Further evidence of GHRH gene expression in the ovary and testis was provided by reverse transcription polymerase chain reaction of ovarian mRNA...

Susan Berry C H Srivastava L R Rubin Ora Hirsch Pescovitz

Although the sequence of human GH-releasing hormone (GHRH) has been determined, all of the information concerning gene expression has been based on pathological sources of ectopic GHRH, since the only established physiological source of GHRH in humans is the hypothalamus. We recently reported the presence of extrahypothalamic GHRH-like mRNA and immunoreactive material in rat testis and placenta. To determine if human testis and placenta also contain immunoreactive GHRH-like peptides, tissue extracts were analyzed using enzyme-linked immunosorbent assay methodology. Both tissues had detectable quantities of immunoreactive peptide recognized by a monoclonal antibody to synthetic human GHRH-(1-44) (testis, 2.4 ng/g tissue, 0.68 ng/mg protein; placenta, 2.6 ng/g tissue, 0.36 ng/mg protein). The origin of these peptides was confirmed by extracting total RNA from human testis and placenta, with analysis on Northern blots probed with riboprobes for rat hypothalamic GHRH cDNA and human pancreatic tumor GHRH cDNA. Human testis and placenta total RNA both contain an approximately 790-nucleotide RNA species similar in size to that reported in ectopic GHRH-producing human tumors. In addition, two larger hybridization signals were seen at 3000 and 4900 nucleotides. These data suggest that testis and placenta are extrahypothalamic sites of expression of the human GHRH gene. Normal expression of the GHRH gene in extrahypothalamic sites may include transcription of larger mRNA species than those observed in ectopic pathological sources of GHRH expression.

K Hashimoto

Masafumi Koga Takashi Motomura T Kishimoto

The expression of GHRH receptor (GHRH-R) messenger ribonucleic acid (mRNA) was studied in 22 pituitary adenomas and 2 normal anterior pituitaries. Northern blot analysis revealed that GHRH-R mRNA were expressed in all 14 GH-producing adenomas, 1 of 3 ACTH-producing adenomas, the 1 PRL-producing adenoma, 2 of 4 nonfunctioning adenomas, and the 2 normal anterior pituitaries. Their expression levels varied among GH-producing adenomas and were relatively low in GH-nonproducing adenomas. In addition to the major transcript with a molecular mass of 2.0 kilobases (kb), the transcripts were identified at 2.8 and 4.5 kb in some GH-producing adenomas. To examine the structural variations in GHRH-R mRNA in pituitary adenomas, we amplified the complementary DNA fragment encompassing the region from the third cytoplasmic loop to the sixth transmembrane domain of GHRH-R. This region was selected because this region of the G protein-coupled receptor has been known to interact with G protein. Two amplified fragments with...

Kelly E. Mayo

Teresa Miller

Venita Dealmeida Shane R Cunha

Hormones from the hypothalamus mediate interactions between the nervous and endocrine systems by controlling the activity of specific target cells in the anterior pituitary gland. The hypothalamic peptide, growth hormone-releasing hormone (GHRH), acts on pituitary somatotroph cells to stimulate their proliferation during development and to regulate their ability to produce and secrete growth hormone (GH). These actions are mediated by a recently identified receptor for GHRH that belongs to family B-III of the G protein-coupled receptor superfamily. The rat GHRH receptor is expressed predominantly in the pituitary gland and in somatotroph cells. To investigate this tissue- and cell-specific expression, the receptor gene has been cloned and characterized. The receptor gene promoter is selectively expressed in pituitary cells and is regulated by the pituitary-specific transcription factor Pit-1. There is a sexual dimorphism in GHRH receptor expression in the rat pituitary. suggesting regulation by gonadal steroids. In addition, glucocorticoids are potent positive regulators of GHRH receptor gene expression. Substantial evidence points to an important role for GHRH in regulating the proliferation and functional activity of the somatotroph cell. This ih best observed in the dwarf little mouse, which harbors a mutation in the extracellular domain of the GHRH receptor that abolishes the receptor's hormone-binding and signaling properties, resulting in severe somatotroph hypoplasia. Complementary studies in transgenic mice overexpressing the ligand GHRH reveal corresponding somatotroph hyperplasia. Consistent with these observations, GHRH potently activates the MAP kinase pathway in pituitary somatotroph cells. To better understand the hormone-binding and signaling properties of the GHRH receptor. mutant and chimeric receptors have been analyzed to define domains important for GHRH interaction. The GHRH receptor signals predominantly through cAMP-dependent pathways; however, a variant form of the GHRH receptor with an insertion into the third intracellular domain, generated through alternative RNA processing, binds GHRH but fails to signal, suggesting potential modulation of receptor function at a post-transcriptional level. This chapter will integrate these basic investigations of GHRH and its receptor with current information on the involvement of the GHRH signaling system in human diseases of GH secretion and growth.

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