Human Growth Hormone-releasing Hormone Receptor Research Articles

Constitutive signal bias mediated by the human GHRHR splice variant 1

Alternative splicing of G protein-coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone-releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to β-arrestins. Based on the cryogenic electron microscopy structures of SV1 in the apo state or GHRH-bound state in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs versus β-arrestins. As suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias toward β-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

Antagonists of Growth Hormone-Releasing Hormone Inhibit the Growth of Pituitary Adenoma Cells by Hampering Oncogenic Pathways and Promoting Apoptotic Signaling.

Simple SummaryMany studies have demonstrated that the antagonists of growth hormone-releasing hormone (GHRH) exert inhibitory activities in a variety of experimental cancers; however, their potential antitumor role in pituitary adenomas (PAs) remains largely unknown. Here, we show that GHRH antagonists of Miami (MIA) class, MIA-602 and MIA-690, are able to reduce the growth and promote cell death in hormone-secreting PA cell lines, through the inhibition of mechanisms implicated in tumorigenesis and cancer progression. MIA-602 and MIA-690 also decreased the viability of tumor cells derived from human pituitary tumors. Overall, these findings suggest that GHRH antagonists may represent new therapeutic tools for the treatment of PAs, both alone or in combination with standard pharmacological treatments.Pituitary adenomas (PAs) are intracranial tumors, often associated with excessive hormonal secretion and severe comorbidities. Some patients are resistant to medical therapies; therefore, novel treatment options are needed. Antagonists of growth hormone-releasing hormone (GHRH) exert potent anticancer effects, and early GHRH antagonists were found to inhibit GHRH-induced secretion of pituitary GH in vitro and in vivo. However, the antitumor role of GHRH antagonists in PAs is largely unknown. Here, we show that the GHRH antagonists of MIAMI class, MIA-602 and MIA-690, inhibited cell viability and growth and promoted apoptosis in GH/prolactin-secreting GH3 PA cells transfected with human GHRH receptor (GH3-GHRHR), and in adrenocorticotropic hormone ACTH-secreting AtT20 PA cells. GHRH antagonists also reduced the expression of proteins involved in tumorigenesis and cancer progression, upregulated proapoptotic molecules, and lowered GHRH receptor levels. The combination of MIA-690 with temozolomide synergistically blunted the viability of GH3-GHRHR and AtT20 cells. Moreover, MIA-690 reduced both basal and GHRH-induced secretion of GH and intracellular cAMP levels. Finally, GHRH antagonists inhibited cell viability in human primary GH- and ACTH-PA cell cultures. Overall, our results suggest that GHRH antagonists, either alone or in combination with pharmacological treatments, may be considered for further development as therapy for PAs.

Rescue of Isolated GH Deficiency Type II (IGHD II) via Pharmacologic Modulation of GH-1 Splicing.

Isolated GH deficiency (IGHD) type II, the autosomal dominant form of GHD, is mainly caused by mutations that affect splicing of GH-1. When misspliced RNA is translated, it produces a toxic 17.5-kDa GH isoform that reduces the accumulation and secretion of wild-type-human GH (wt-hGH). Usually, isolated GHD type II patients are treated with daily injections of recombinant human GH in order to maintain normal growth. However, this type of replacement therapy does not prevent toxic effects of the 17.5-kDa GH isoform on the pituitary gland, which can eventually lead to other hormonal deficiencies. Here, we tested the possibility to restore the constitutive splicing pattern of GH-1 by using butyrate, a drug that mainly acts as histone deacetylase inhibitor. To this aim, wt-hGH and/or different hGH-splice site mutants (GH-IVS3+2, GH-IVS3+6, and GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GHRHR) (GC-GHRHR). Upon butyrate treatment, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed increased GH transcript level, intracellular GH content, and GH secretion when compared with the corresponding untreated condition. The effect of butyrate was most likely mediated by the alternative splicing factor/splicing factor 2. Overexpression of alternative ASF/SF2 in the same experimental setting, indeed, promoted the amount of full-length transcripts thus increasing synthesis and secretion of the 22-kDa GH isoform. In conclusion, our results support the hypothesis that modulation of GH-1 splicing pattern to increase the 22-kDa GH isoform levels can be clinically beneficial and hence a crucial challenge in GHD research.

Abstract 4769: Growth hormone-releasing hormone (GHRH) antagonist attenuates cell motility of human endometrial cancer by down-regulating Twist and N-Cadherin expression.

Abstract Introduction : More than 25% of patients diagnosed with endometrial carcinoma have an invasive primary cancer accompanied by metastases. GHRH is secreted by the hypothalamus and stimulates the synthesis and secretion of GH from the pituitary. The expression of GHRH and its receptors has been demonstrated in peripheral tissues. In recent years, many antagonistic analogs of GHRH were developed. GHRH antagonists have effects on tumor cells, but the underlying molecular mechanism is not well known. Although Twist and N-Cadherin play important roles in cancer cell motility, it is not established whether Twist and N-Cadherin are required for GHRH antagonists-attenuated cell motility on endometrial cancer cells. In the present study, we examined the action of GHRH antagonist-attenuated cell motility and the mechanisms of the action in endometrial cancer. Methods and Materials : Endometrial cancer cell line Ishikawa and ECC-1 were derived from an endometrial adenocarcinoma. GHRH antagonist MIA-602 was synthesized by solid phase methods. Cell motility was estimated by invasion and migration assay. Immunoblot analysis and RT-PCR were performed to investigate the expression of GHRH receptor, GHRH, and the effects of GHRH antagonist in Twist and N-Cadherin. Human GHRH receptor siRNA was used to knock down the expression of GHRH receptor for elucidating the mechanisms of GHRH antagonist action. Results : The GHRH receptor was expressed in human endometrial cancer cells. The GHRH antagonist attenuated cell motility in a dose-dependent manner. GHRH antagonist-attenuated cell motility was restored in cells pretreated with GHRH receptor siRNA. GHRH antagonist suppressed Twist and N-Cadherin signaling. Knockdown of GHRH receptor with siRNA recovered the expression of Twist and N-Cadherin signaling. Conclusions These results demonstrate that the GHRH antagonist suppressed the cell motility of human endometrial cancer through the GHRH receptor and the down-regulation of Twist and N-Cadherin. Our findings represent a new concept regarding the mechanisms of GHRH antagonist-suppressed cell motility in human endometrial cancer, suggesting the possibility of GHRH antagonist as a potential therapeutic intervention for the treatment of human endometrial cancer. Citation Format: Hsien-Ming Wu, Andrew V. Schally, Peter C.K. Leung. Growth hormone-releasing hormone (GHRH) antagonist attenuates cell motility of human endometrial cancer by down-regulating Twist and N-Cadherin expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4769. doi:10.1158/1538-7445.AM2013-4769 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Interaction of PICK1 with C-Terminus of Growth Hormone–Releasing Hormone Receptor (GHRHR) Modulates Trafficking and Signal Transduction of Human GHRHR

Release of growth hormone (GH) from the somatotroph is regulated by binding GH-releasing hormone (GHRH) to its cognate receptor (GHRHR), one of the members of the G protein–coupled receptor (GPCR) superfamily. Proteins bound to the carboxy (C)-terminus of GPCR have been reported to regulate intracellular trafficking and function of the receptor; however, no functionally significant protein associated with GHRHR has been reported. We have identified a protein interacting with C-kinase 1 (PICK1) as a binding partner of GHRHR. In vitro binding assay revealed the PDZ-domain of PICK1 and the last four amino acid residues of GHRHR were prerequisite for the interaction. Further, in vivo association of these proteins was confirmed. Immunostaining data of a stable cell line expressing GHRHR with or without PICK1 suggested the C-terminus of GHRHR promoted cell surface expression of GHRHR and PICK1 affected the kinetics of the cell surface expression of GHRHR. Furthermore, cAMP production assay showed the C-terminus of GHRHR is involved in the regulation of receptor activation, and the interaction of GHRHR with PICK1 may influence intensities of the signal response after ligand stimulation. Thus, the interaction of the C-terminus of GHRHR with PICK1 has a profound role in regulating the trafficking and the signaling of GHRHR. [Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.12287FP]

Identification and functional analysis of novel human growth hormone-releasing hormone receptor (GHRHR) gene mutations in Japanese subjects with short stature

Growth hormone-releasing hormone receptor (GHRHR) gene mutations have been identified in patients of different ethnic origins with isolated GH deficiency (IGHD) type IB. However, the prevalence of these mutations in the Japanese population has yet to be fully determined. This study aimed to evaluate the contributions of GHRHR mutations to the molecular mechanism underlying short stature in Japanese subjects. The GHRHR gene was sequenced in 127 unrelated Japanese patients with either IGHD (n = 14) or idiopathic short stature (ISS; n = 113). Sequence variants were evaluated in family members and 188 controls, and then examined in functional studies. A novel homozygous E382E (c.1146G>A) synonymous variant, at the last base of exon 12, was identified in an IGHD family with two affected sisters. In vitro splicing studies showed this mutation to result in skipping of exon 12. In one ISS patient, a heterozygous ATG-166T>C variant was found in the distal Pit-1 P2 binding element of the GHRHR promoter. In two control subjects, a close but distinct variant, ATG-164T>C, was detected. Functional studies showed that both promoter variants diminish promoter activity by altering Pit-1 binding ability. Four missense variants were also found in both patient and control groups but had no detectable functional consequences. The homozygous GHRHR mutation was rare, being detected in only one Japanese IGHD family. Future research is needed to clarify the genetic contributions of heterozygous functional promoter variants to GHD, ISS and normal-stature variations.

Growth Hormone (GH)-Releasing Hormone Increases the Expression of the Dominant-Negative GH Isoform in Cases of Isolated GH Deficiency due to GH Splice-Site Mutations

An autosomal dominant form of isolated GH deficiency (IGHD II) can result from heterozygous splice site mutations that weaken recognition of exon 3 leading to aberrant splicing of GH-1 transcripts and production of a dominant-negative 17.5-kDa GH isoform. Previous studies suggested that the extent of missplicing varies with different mutations and the level of GH expression and/or secretion. To study this, wt-hGH and/or different hGH-splice site mutants (GH-IVS+2, GH-IVS+6, GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GC-GHRHR). Upon GHRH stimulation, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed reduced hGH secretion and intracellular GH content when compared with cells expressing only wt-hGH, confirming the dominant-negative effect of 17.5-kDa isoform on the secretion of 22-kDa GH. Furthermore, increased amount of 17.5-kDa isoform produced after GHRH stimulation in cells expressing GH-splice site mutants reduced production of endogenous rat GH, which was not observed after GHRH-induced increase in wt-hGH. In conclusion, our results support the hypothesis that after GHRH stimulation, the severity of IGHD II depends on the position of splice site mutation leading to the production of increasing amounts of 17.5-kDa protein, which reduces the storage and secretion of wt-GH in the most severely affected cases. Due to the absence of GH and IGF-I-negative feedback in IGHD II, a chronic up-regulation of GHRH would lead to an increased stimulatory drive to somatotrophs to produce more 17.5-kDa GH from the severest mutant alleles, thereby accelerating autodestruction of somatotrophs in a vicious cycle.

Naturally-occurring missense mutations in the human growth hormone-releasing hormone receptor alter ligand binding

Growth hormone (GH) releasing hormone (GHRH) is a hypothalamic factor that stimulates GH secretion. It acts by activating a seven transmembrane domain G protein-coupled receptor of 423 amino acids expressed by the somatotroph cells of the pituitary gland (GHRH receptor, GHRH-R). Familial isolated growth hormone deficiency (IGHD) can be caused by mutations in the GHRH-R gene both in humans and mice. We have described six disease-causing missense mutations in this gene in IGHD patients (H137L, L144H, A176V, A222E, F242C, K329E). These mutations are inherited as autosomal recessive traits, and cause impairment of the receptor to transmit GHRH signalling. The aim of this study is to investigate the mechanisms through which these mutations cause receptor malfunction. To this end, we transiently expressed each mutated receptor into Chinese hamster ovary cells. Cells expressing each of the mutated receptors did not show an increase in intracellular cyclic AMP in response to GHRH. Immunoprecipitation and immunofluorescence studies indicated that the amino acid changes do not cause protein degradation, and do not alter the proper insertion of the receptor into the cell membrane. Binding studies with human 125I-GHRH showed that the lack of response to GHRH is due to inability of all the mutated receptors to bind the ligand. These studies demonstrate that abnormal ligand binding is a common mechanism by which naturally occurring missense mutation alter GHRH-R function.

Changes in Somatostatin Receptor mRNA Levels by G Protein Mutation in GH3 Cells Which Show Responsiveness to Growth Hormone-Releasing Hormone

Background:GH3 cells lack growth hormone(GH)-releasing hormone(GHRH) receptors. In this study, GH3 cells permanently transfected with human GHRH receptor cDNA(GH3-GHRHR cells), were established in order to examine the effects of GHRH and G protein mutation(gsp oncogene) on the levels of somatostatin receptor mRNA. Methods:GH3 cells were permanently transfected with a plasmid expressing human GHRH receptor cDNA. The GHRH receptor mRNA was detected by RT-PCR. The responsiveness to GHRH was evaluated using a GHRH binding assay, Western blot analysis, Northern blot analysis, and measurements of the intracellular cAMP levels and GH release. Cells were transiently transfected with the gsp oncogene, and then treated with GHRH or octreotide for 4h. The sst1 and sst2 mRNA levels were measured using real-time RT-PCR analyses. Results:GHRH receptor mRNA was detected in the GH3 cells permanently transfected with human GHRH receptor cDNA. The GHRH binding assay showed that GHRH was bound to the GH3-GHRHR cells. The GHRH treatment increased the intracellular cAMP levels, GH release, GH mRNA levels, and MAPK activity, as well as the levels of sst1 and sst2 mRNA. Transient expression of the gsp oncogene for 48h increased the cAMP, GH release, and levels of sst1 and sst2 mRNA. In the gsp-transfected GH3-GHRHR cells, GHRH stimulation resulted in decreases in the magnitude of the increase in the levels of sst1 and sst2 mRNA compared to those transfected with a control vector. Octreotide treatment did not alter the levels of sst1 and sst2 mRNA in either the control or gsp-transfected cells. Conclusions:These results suggest that GH3 cells permanently transfected with the GHRH receptor are useful in the in vitro studies on the actions of GHRH. The gsp oncogene was shown to increases the levels of sst1 and sst2 mRNA in GH3 cells, but these findings are unlikely to be the major mechanism by which gsp-positive pituitary tumors show a greater response to somatostatin. The discrepancy between the in vivo and these in vitro results should be examined further.

Familial Growth Hormone Deficiency and Mutations in the GHRH Receptor Gene

Growth hormone (GH)-releasing hormone (GHRH) is necessary for the proliferation of the somatotropic cells of the anterior pituitary and the synthesis and secretion of GH. GHRH is released by the hypothalamus into the portal hypophysial circulation to bind to a membrane surface receptor [GHRH receptor (GHRHR)] expressed by the somatotropic cells. Because of the need of GHRH for GH secretion, it is to be expected that alterations in synthesis or action of GHRH would result in isolated GH deficiency (IGHD). Indeed, although GHRH gene mutations have never been reported, mutations in the GHRHR gene (GHRHR) are emerging as a relatively common cause of inherited autosomal recessive IGHD. The first human GHRHR mutations were discovered in families with a history of parental consanguinity. More recently, kindreds in which IGHD subjects are compound heterozygotes for two distinct mutations indicate that faulty GHRHR alleles may be prevalent and that these mutations may need to be suspected even in sporadic IGHD cases. Patients with two faulty GHRHR alleles have normal weight at birth. Growth failure becomes apparent during the first year of life. Biochemical studies show low serum insulin-like growth factor-1 level, and absent or markedly reduced GH response to a variety of stimuli. Magnetic resonance imaging shows hypoplasia of the anterior pituitary. In this chapter, we describe the GHRHR mutations reported to date and the phenotype of affected individuals.

Inhibitory effects of antagonistic analogs of GHRH on GH3 pituitary cells overexpressing the human GHRH receptor.

GH3 rat pituitary tumor cells produce GH and prolactin (PRL), but lack the GHRH receptor (GHRH-R). We expressed human GHRH-R (hGHRH-R) in GH3 cells using recombinant adenoviral vectors and studied the effects of GHRH antagonists. The mRNA expression of the GHRH-R gene in the cells was demonstrated by RT-PCR. An exposure of the GH3 cells infected with hGHRH-R to 10(-10), 10(-9) and 10(-8) m hGHRH for 1 or 2 h in culture caused a dose-dependent elevation of the intracellular cAMP concentration and the cAMP efflux. Exposure to hGHRH also elicited dose-dependent increases in GH and PRL secretion from these cells. Neither the uninfected nor the antisense hGHRH-R-infected control cells exhibited cAMP, GH and PRL responses to GHRH stimulation. GHRH antagonists JV-1-38 and jv-1-36 applied at 3x10(-8) m for 3 h, together with 10(-9) m GHRH, significantly inhibited the GHRH-stimulated cAMP efflux from the hGHRH-R-infected cells by 36 and 80% respectively. The more potent antagonist JV-1-36 also decreased the intracellular cAMP levels in these cells by 55%. Exposure to JV-1-36 for 1 h nullified the stimulatory effect of GHRH on GH secretion and significantly inhibited it by 64 and 77% after 2 and 3 h respectively. In a superfusion system, GHRH at 10(-10), 10(-9) and 10(-8) m concentrations induced prompt and dose-related high cAMP responses and smaller increases in the spontaneous GH secretion of the hGHRH-R-infected cells. Antagonists JV-1-36 and JV-1-38 applied at 3x10(-8) m for 15 min, together with 10(-9) m GHRH, inhibited the GHRH-stimulated cAMP response by 59 and 35% respectively. This work demonstrates that GHRH antagonists can effectively inhibit the actions of GHRH on the hGHRH-R. Our results support the view that this class of compounds would be active clinically.

The growth hormone-releasing hormone receptor: desensitisation following short-term agonist exposure.

We have investigated the effect of short-term preexposure of growth hormone-releasing hormone (GHRH) on the subsequent response to GHRH in a baby hamster kidney (BHK) cell line expressing the human GHRH receptor and in primary rat pituitary cells. In the BHK cells the receptor was rapidly desensitised in a homologous fashion. Preexposure with agents directly stimulating the cAMP pathway like forskolin and db-cAMP had no effect. In rat pituitary cells we also observed a rapid desensitisation of the GHRH response in an apparently homologous fashion. In both systems the desensitisation was dose-dependent with no change in the potency of the hormone in a subsequent stimulation, only the efficacy was decreased. In the rat pituitary cell, the response measured as growth hormone release was more sensitive to the agonist-induced desensitisation than the cAMP response. No indication of depletion of growth hormone (GH) stores was seen. In rat pituitary cells, contrary to observations in BHK cells, preexposure with both forskolin and db-cAMP desensitised a subsequent growth hormone-releasing hormone stimulation, indicating a heterologous desensitisation. Phorbol-12-myristate 13-acetate (PMA), on the other hand, had no effect. In the baby hamster kidney cells it was demonstrated that the GHRH receptor surface expression decreased following preexposure with GHRH. This phenomenon was observed only in whole cells suggesting a rapid internalisation process. Together, these data indicate that after short-term GHRH preexposure, both in a human and rat system, the following GHRH response is desensitised. In BHK cells this desensitisation is strictly homologous. In rat pituitary cells, on the other hand, the desensitisation is a mixed homologous/ heterologous type.

The Growth Hormone-Releasing Hormone Receptor: Desensitisation Following Short-Term Agonist Exposure*

We have investigated the effect of short-term preexposure of growth hormone-releasing hormone (GHRH) on the subsequent response to GHRH in a baby hamster kidney (BHK) cell line expressing the human GHRH receptor and in primary rat pituitary cells. In the BHK cells the receptor was rapidly desensitised in a homologous fashion. Preexposure with agents directly stimulating the cAMP pathway like forskolin and db-cAMP had no effect. In rat pituitary cells we also observed a rapid desensitisation of the GHRH response in an apparently homologous fashion. In both systems the desensitisation was dose-dependent with no change in the potency of the hormone in a subsequent stimulation, only the efficacy was decreased. In the rat pituitary cell, the response measured as growth hormone release was more sensitive to the agonist-induced desensitisation than the cAMP response. No indication of depletion of growth hormone (GH) stores was seen. In rat pituitary cells, contrary to observations in BHK cells, preexposure with both forskolin and db-cAMP desensitised a subsequent growth hormone-releasing hormone stimulation, indicating a heterologous desensitisation. Phorbol-12-myristate 13-acetate (PMA), on the other hand, had no effect. In the baby hamster kidney cells it was demonstrated that the GHRH receptor surface expression decreased following preexposure with GHRH. This phenomenon was observed only in whole cells suggesting a rapid internalisation process. Together, these data indicate that after short-term GHRH preexposure, both in a human and rat system, the following GHRH response is desensitised. In BHK cells this desensitisation is strictly homologous. In rat pituitary cells, on the other hand, the desensitisation is a mixed homologous/ heterologous type.

Restoration of growth hormone-releasing hormone (GHRH) responsiveness in pituitary GH3 cells by adenovirus-directed expression of the human GHRH receptor.

GH-secreting GH3 cells lack GH-releasing hormone (GHRH) receptors. In this study we used adenoviral vectors to transfer the human GHRH receptor to GH3 cells in an effort to restore GHRH responsiveness. A replication-deficient recombinant adenovirus (AdGHRH-R) was designed to allow cytomegalovirus promoter-driven expression of the GHRH receptor messenger RNA. COS-7 cells and GH-producing GH3 cells infected with AdGHRH-R showed GHRH receptor expression on their membranes and exhibited specific GHRH binding. The addition of GHRH to GH3 cells infected with AdGHRH-R increased cAMP levels, induced cAMP response element-binding protein phosphorylation and restored GH secretory responsiveness. GHRH treatment also caused activation of mitogen-activated-protein kinase, induction of c-fos, stimulation of GH promotor activity, and increased cellular proliferation. These findings indicate that adenoviral vectors carrying human GHRH receptor are useful for in vitro studies of GHRH receptor biology and represent a first step toward the development of gene therapy for dwarfism caused by GHRH receptor mutations.

Restoration of Growth Hormone-Releasing Hormone (GHRH) Responsiveness in Pituitary GH3 Cells by Adenovirus-Directed Expression of the Human GHRH Receptor

GH-secreting GH3 cells lack GH-releasing hormone (GHRH) receptors. In this study we used adenoviral vectors to transfer the human GHRH receptor to GH3 cells in an effort to restore GHRH responsiveness. A replication-deficient recombinant adenovirus (AdGHRH-R) was designed to allow cytomegalovirus promoter-driven expression of the GHRH receptor messenger RNA. COS-7 cells and GH-producing GH3 cells infected with AdGHRH-R showed GHRH receptor expression on their membranes and exhibited specific GHRH binding. The addition of GHRH to GH3 cells infected with AdGHRH-R increased cAMP levels, induced cAMP response element-binding protein phosphorylation and restored GH secretory responsiveness. GHRH treatment also caused activation of mitogen-activated-protein kinase, induction of c-fos, stimulation of GH promotor activity, and increased cellular proliferation. These findings indicate that adenoviral vectors carrying human GHRH receptor are useful for in vitro studies of GHRH receptor biology and represent a first step toward the development of gene therapy for dwarfism caused by GHRH receptor mutations.

Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers.

The proliferation of various tumors is inhibited by the antagonists of growth hormone-releasing hormone (GHRH) in vitro and in vivo, but the receptors mediating the effects of GHRH antagonists have not been identified so far. Using an approach based on PCR, we detected two major splice variants (SVs) of mRNA for human GHRH receptor (GHRH-R) in human cancer cell lines, including LNCaP prostatic, MiaPaCa-2 pancreatic, MDA-MB-468 breast, OV-1063 ovarian, and H-69 small-cell lung carcinomas. In addition, high-affinity, low-capacity binding sites for GHRH antagonists were found on the membranes of cancer cell lines such as MiaPaCa-2 that are negative for the vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptor (VPAC-R) or lines such as LNCaP that are positive for VPAC-R. Sequence analysis of cDNAs revealed that the first three exons in SV(1) and SV(2) are replaced by a fragment of retained intron 3 having a new putative in-frame start codon. The rest of the coding region of SV(1) is identical to that of human pituitary GHRH-R, whereas in SV(2) exon 7 is spliced out, resulting in a 1-nt upstream frameshift, which leads to a premature stop codon in exon 8. The intronic sequence may encode a distinct 25-aa fragment of the N-terminal extracellular domain, which could serve as a proposed signal peptide. The continuation of the deduced protein sequence coded by exons 4-13 in SV(1) is identical to that of pituitary GHRH-R. SV(2) may encode a GHRH-R isoform truncated after the second transmembrane domain. Thus SVs of GHRH-Rs have now been identified in human extrapituitary cells. The findings support the view that distinct receptors are expressed on human cancer cells, which may mediate the antiproliferative effect of GHRH antagonists.

Structure and function of the growth-hormone-releasing hormone receptor

Growth-hormone-releasing hormone (GHRH) stimulates growth hormone (GH) secretion and GH synthesis and is also thought to cause somatotroph proliferation. Specific high-affinity binding sites for GHRH have been demonstrated on pituitary membranes using iodinated GHRH analogs. The complementary DNA encoding for the human GHRH receptor (GHRH-R) was recently cloned. The open reading frame was shown to extend 1269 bp and thus to encode a protein of 423 amino acids with a predicted molecular weight of 47 kDa. Expression is restricted to specific tissues. Analysis of the genomic structure revealed that the human GHRH-R gene spans 15 kb and consists of 13 exons. The 5'-flanking region of the human GHRH-R gene was recently characterized. Transcriptional regulation of the GHRH-R is discussed in this review. Mechanisms of signal transduction for control of GH transcription and secretion are presented. Furthermore, the role of the GHRH-R in proliferation and differentiation of the somatotrophic pituitary cell as well as in disease is examined.

Restricted Presence of the Growth Hormone-Releasing Hormone Receptor to Somatotropes in Rat and Human Pituitaries

Specific binding of growth hormone-releasing hormone (GHRH) to its plasma membrane receptor represents the first step of cellular signals leading to exocytotic GH secretion in the anterior pituitary. The GHRH receptor (GHRH-R) has been cloned and belongs to the secretin/glucagon/vasoactive intestinal peptide subfamilly of G-protein-coupled receptors. To study its characteristics in rat and human pituitaries and examine its cellular and subcellular localization, a site-directed polyclonal antibody recognizing the C-terminal portion 392–404 of the rat and human GHRH-R was used. Immunohistochemistry was performed on paraffin-embedded pituitary sections while ultrastructural immunocytology was done on frozen and Lowicryl-resin-embedded ultrathin sections. GHRH-R-like immunoreactivity was restricted to somatotropes and colocalized with GH in both rat and human tissues. No signal was detected in gonadotropes, lactotropes, corticotropes and thyrotropes. At the subcellular level, gold particles were associated with the plasma membrane (observed on ultrathin frozen sections), secretory granule membrane, cytoplasmic matrix, nuclear membrane and nuclear matrix. In the nucleus, gold particles were mainly observed at the junction between eu- and heterochromatin. The highest density of labeling was observed in the cytoplasm (55 vs. 45% in the nucleus), mainly in secretory granules (59% of cytoplasmic labeling) and the plasma membrane. These results support the hypothesis that GHRH-mediated actions in the pituitary are specific to somatotropes and that GHRH-R isoforms and/or ligand-receptor complexes are involved in intracellular trafficking, recycling processes and nuclear functions.

Cloning and Characterization of the 5′-Flanking Region of the Human Growth Hormone-releasing Hormone Receptor Gene

We cloned the 5'-flanking region of the human growth hormone-releasing hormone receptor (GHRH-R) gene and determined the nucleotide sequence of 2.7 kilobases upstream from the translation start site. RNase protection analysis showed the major transcription start site is 122 base pairs upstream from the translation start site. The 5'-end of the longest product of 5'-rapid amplification of cDNA ends was close to the site. There were no typical TATA homologies but several putative regulatory elements including Pit-1-binding site-like element. Transient transfection studies using a luciferase reporter gene demonstrated that 5'-flanking region had promoter activity in GH3 cells (derived from rat pituitary tumor) but not in nonpituitary cells, BeWo and HeLa cells. However, co-transfection of Pit-1 expression vector increased luciferase activity in BeWo cells. Deletion study showed that the regions from -310 to -130 and from -130 to -120 were important for the GHRH-R gene expression in GH3 cells, although the latter contributed less to the gene expression. In BeWo cells co-transfected with Pit-1 expression vector, the region from -310 to -130 was essential for the Pit-1-dependent expression of GHRH-R gene. The region from -310 to -120 has two putative Pit-1-binding sites, P1 and P2, located from -129 to -123 and from -171 to -160, respectively. Both mobility shift assay and DNase-I footprint analysis showed that P2 had much higher Pit-1 binding affinity than P1. Mutation of P2 decreased GHRH-R gene expression in GH3 cells. These findings were consistent with the results that the region from -310 to -130 is an important element for Pit-1-dependent expression of GHRH-R gene.

Significance of growth hormone-releasing hormone receptor mRNA in non-neoplastic pituitary and pituitary adenomas: a study by RT-PCR and in situ hybridization.

This study examined the expression of human growth hormone-releasing hormone receptor (GHRH-R) mRNA in both non-neoplastic pituitary tissues and pituitary adenomas by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). RT-PCR analysis showed that all of the non-neoplastic pituitaries and all GH-producing adenomas, one prolactinoma and one third of the non-functioning adenomas expressed GHRH-R mRNA. ISH demonstrated that all of GH-producing adenomas and two prolactinomas expressed GHRH-R mRNA. The expression of GHRH-R mRNA in GH-producing adenomas was greater than that in the other adenomas by RT-PCR and ISH. GHRH-R mRNA detected by ISH was observed only in GH cells from the pituitary gland of a young girl. In pituitary adenomas, a diffuse signal was observed in the cytoplasm of all of the GH-producing adenomas and in two prolactinomas. Expression of GHRH-R mRNA was not seen in normal prolactin cells, or in any adenomas other than GH-producing adenomas and a few prolactinomas. These results suggest that GHRH-R mRNA plays a role mainly in the function of GH-producing adenomas but may also play a role in function of some prolactinomas.