Molecular Cloning and Expression of a Pituitary-Specific Receptor for Growth Hormone-Releasing Hormone

Kelly

E. Mayo

Department of Biochemistry, Northwestern University Evanston, Illinois 60208

Molecular

Biology,

and Cell Biology

A novel cDNA was isolated from rat pituitary mRNA using the polymerase chain reaction to amplify sequences encoding G protein-coupled receptors. The human homolog of this cDNA was isolated and expressed in human kidney 293 cells, and membrane fractions from these cells were found to bind human GH-releasing hormone (GHRH) with high affinity and specificity. GHRH also stimulates intracellular CAMP production in these transfected cells. The encoded receptor protein contains seven potential membrane-spanning domains, a hallmark of G proteincoupled receptors, and is homologous to previously identified receptors for secretin and vasoactive intestinal peptide, ligands that are related to GHRH. The rat GHRH receptor mRNA is expressed predominantly, if not exclusively, in the anterior pituitary gland, the major target for GHRH action. These results define a mechanism for cellular signaling by GHRH and provide the opportunity to examine the role of the GHRH receptor in growth abnormalities that involve the GH axis. (Molecular Endocrinology 6: 1734-1744,1992)

member of the cytokine receptor family, and signal transduction by this receptor is not well understood (10). Defects in the GH receptor in patients with Laron dwarfism demonstrate its important role in growth (11). Last, two recently identified somatostatin receptors (7) have the seven membrane-spanning domains characteristic of receptors that are coupled to intracellular signaling pathways by G proteins (12). GHRH (also referred to as GRF) is a peptide hormone (42-44 amino acids in various species) that is synthesized in the hypothalamus and stimulates the secretion of GH from pituitary somatotropes (13-I 6). GHRH belongs to a growing family of peptides which includes glucagon, vasoactive intestinal peptide (VIP), secretin, gastric inhibitory peptide (GIP), peptide with histidine as N-terminus and isoleucine as C-terminus (PHI), and pituitary adenylate cyclase activating peptide (PACAP) (16-18). Specific high affinity binding sites for GHRH have been measured on pituitary membranes using several different iodinated GHRH analogs (1 g-21) and GHRH binding proteins ranging in size from 26-72 kilodaltons have been identified by chemical crosslinking with anterior pituitary cells (22, 23). GHRH binding to pituitary cells results in the activation of adenylate cyclase, and guanine nucleotides inhibit GHRH binding, suggesting that Gs is an intermediate in GHRH action (24-26). Consistent with the notion that CAMP is an important second messenger for GHRH signaling, both GHRH and CAMP increase pituitary GH gene expression, induce the expression of the protooncogene cfos, and stimulate the proliferation of pituitary somatotropic cells (27-30). Several observations suggest that the GHRH receptor, in addition to playing a key role in normal growth regulation, might be important in disorders involving aberrant GH secretion. The presumed target for GHRH receptor action in pituitary somatotrophs, the Gsa protein, has been found to be mutated in some GH-secreting pituitary adenomas associated with altered adenylate cyclase activity (31, 32) suggesting the possibility that the GHRH receptor itself could be a protooncogene subject to activating mutations in some pituitary tumors (33). Alterations in the GHRH receptor-Gs-adenylate cyclase system have also been implicated in the growth deficiencies observed in several dwarf rodent strains

INTRODUCTION The growth of vertebrate organisms is regulated in part by a complex cascade of hormones, including the neuroendocrine peptides GH-releasing hormone (GHRH) and somatostatin, the pituitary protein GH, and the insulin-like growth factors (IGFs) or somatomedins (l3). The actions of these hormones are mediated by specific cell-surface receptors, all of which have been identified and cloned with the exception of the receptor for GHRH (4-7). These receptors have diverse structures and signaling mechanisms. The IGF type I receptor, which may function in both IGF-I and IGF-II signaling, is a protein tyrosine kinase, while the IGF type II receptor, which has high affinity for IGF-II, is a multifunctional protein that also serves as a receptor for mannose-8phosphate (8, 9). The GH receptor is a 0888-8809/92/l 734-l 744$03.00/O Molecular Endocmology CopyrIght 0 1992 by The Endocrme

Soctety

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Pituitary GHRH Receptor cDNAs

1735

(34, 35). In particular, the little mutation in the mouse (36), which is a model for human isolated GH deficiency, involves receptor-associated resistance to GHRH (34). To gain insight into the structure of the GHRH receptor, its mechanism of action, and its involvement in growth disorders, I have identified and characterized potential GHRH receptor cDNA clones from rat and human pituitary. These cDNAs encode proteins that have the expected features of G protein-coupled receptors and are related to the recently identified secretin and VIP receptors. The human cDNA, when expressed in human kidney 293 cells, produces a protein that binds GHRH with high affinity and specificity and increases GHRH-dependent CAMP production by these transfected cells. The highly related rat cDNA detects an mRNA that is specifically expressed in the rat anterior pituitary gland, the major target for GHRH action.

RESULTS Isolation

and Analysis

of Receptor

cDNA Clones

The approach used to identify candidate GHRH receptor clones was based on the amplification of G proteincoupled receptor cDNAs using degenerate oligonucleotide primers (37). Primers were designed based on the sequences of a family of structurally related receptors, including the receptors for calcitonin (CT) (38), PTH (39), and secretin (40). The relatedness between the secretin and GHRH ligands suggested that their receptors might also be homologous, and the recent finding that the CT, PTH, and secretin receptors form a subfamily of G protein-coupled receptors (38-40) provided a structural basis for designing primers that might amplify additional family members. Degenerate oligonucleotides encoding portions of membrane-spanning domains 6 and 7 of these receptors were used to amplify cDNA generated from male rat pituitary, the appropriately sized polymerase chain reaction (PCR) products were cloned, and the DNA sequences of several of the clones were determined. One of the clones isolated, RPRG, encoded a peptide with strong homology to the targeted regions of the CT, PTH, and secretin receptors. RPR6 was used to screen human and rat pituitary cDNA libraries, and a human cDNA clone of 1617 bases (HPR3) was identified that contained an open reading frame encoding a 47-kilodalton protein of 423 amino acids (Fig. 1, A and B). Subsequent screening of the rat pituitary library identified three clones (RPR64, RPRll , and RPR20) that together span 1629 bases and encode a 47-kilodalton protein of 423 amino acids that is 82% identical to the human protein (Fig. 2, A and B). HPR3 and RPRC (the composite rat cDNA) both encode proteins with seven hydrophobic potential membrane-spanning domains (Figs. 1C and 2C), suggesting that they might be G protein-coupled receptors. As expected based on the cloning strategy, the predicted protein product of RPRC is related to the some-

what larger CT and PTH receptors (24% and 27% amino acid identity, respectively). Even greater sequence identity was found between RPRC and the rat secretin receptor (35% identity) and the recently identified rat VIP receptor (40% identity) (41). Similar relatedness between the predicted protein product of HPRS, the human cDNA, and the CT, PTH, secretin, and VIP receptors was found. No additional strong nucleic acid or protein homologies were found in searches of the GenBank and Swiss-Prot databases. The amino acid sequences of the HPR3 and RPRC proteins and the rat secretin and VIP receptors are compared in Fig. 3. Predominantly hydrophobic domains predicted to represent membrane-spanning regions of the secretin and VIP receptors are indicated. There are six cysteine residues in the amino-terminal domain that are completely conserved in the RPRC and HPR3 proteins and in the secretin and VIP receptors (Fig. 3). This region is presumed to represent the extracellular ligand-binding domain. A site for potential Nlinked glycosylation is found in the amino-terminal domain of both the RPRC and HPR3 proteins; a second conserved glycosylation site falls within the third potential membrane-spanning domain and is therefore not likely to be used. One of the rat cDNA clones isolated (RPR13; Fig. 2A) was found to contain an additional 123 bases in the coding sequence compared to another rat cDNA (RPRll) or to the human cDNA. This is predicted to insert 41 amino acids into the encoded protein immediately before membrane-spanning domain 6; the sequence of this insert is shown in Fig. 4A. Analysis of rat genomic clones indicates that this insertion point corresponds to an intron-exon boundary, suggesting that alternative RNA processing g?nerates the larger receptor isoform. The presumed splicing patterns by which these alternate products are generated are shown in Fig. 4B. Preliminary analysis of rat pituitary mRNA using reverse transcription-PCR and primers that span the insertion site identified only the shorter form of the transcript; however, it is possible that expression of these mRNA isoforms in the pituitary is hormonally or developmentally regulated. Expression Cells

of the GHRH Receptor

in Transfected

The structural relatedness between the RPRC and HPR3 receptors and the rat secretin and VIP receptors suggested that the ligand for this novel receptor would indeed be a member of the family of peptides that includes secretin, VIP, GHRH, GIP, PHI, PACAP, and glucagon (16-l 8). To examine the ligand-binding characteristics of this putative receptor, the HPR3 cDNA was cloned into the mammalian cell expression vector pcDNA-1 and was used to generate stable lines of human kidney 293 cells by cotransfection with a G418 resistance-selectable marker, pSV2-neo. A clonal line (293-HPR9) that had several copies of the transfected gene and expressed high amounts of receptor mRNA

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MOL 1736

END0.1992

Vo16No.10

HPRJ HPR7

75 150 225 3cQ 315 450 525 600 675 750 825 ml 975 1050 1125 1200 1275 1350 1425 1500 1575 1617

320 240

160 80 0 -80 -160 -240 -320

Fig. 1. Structure of the Human Pituitary GHRH Receptor cDNAs and Protein A, Schematic maps of human GHRH receptor cDNA clones. A composite cDNA showing the location of the 1269-basepair open reading frame (shaded rectangle) is shown. Above this are the two human cDNA clones isolated, along with a partial restriction map of the HPR3 cDNA. 6, DNA sequence and predicted amino acid sequence of human cDNA clone HPR3. C, Hydropathy plot of the human GHRH receptor protein. Seven strongly hydrophobic regions large enough to span the plasma membrane are numbered l-7. The GenBank accession number for the HPR3 cDNA clone is LO1406.

was selected for further analysis. Crude membrane fractions from these cells, or from the control 293 cells (293-WT), were used for binding studies with the ligand [‘251-Tyr’o]human GHRH (hGHRH) (I-44)-amide (19). As shown in Fig. 5A, hGHRH bound to membranes from the transfected cell line 293-HPR9 in a saturable fash-

ion. Scatchard analysis of the binding data, shown in Fig. 5B, revealed a dissociation constant for hGHRH interaction with the receptor in 293-HPR9 cells of approximately 30 PM with approximately 50,000 binding sites per cell. GHRH binding to the receptor in 293HPR9 cells was also examined in competition experi-

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Pituitary

GHRH

Receptor

cDNAs

1737

75 1M 225 300 375 45l 525 6M) 675 750 I325 900 975 LO50 L125

1200 1275

13% 1425

1SM) 1575 1629

C,

1

2

3

4

5

6

7

I

320 240 160 80 0 -80

-160 -240 -320

Fig. 2. Structure of the Rat Pituitary GHRH Receptor cDNAs and Protein A, Schematic maps of rat GHRH receptor cDNA clones. A composite cDNA showing the location of the 1269-basepair open reading frame (shaded rectangle) is shown. Above this are the four rat cDNA clones isolated, along with a partial restriction map of the composite cDNA (RPRC). RPR6 is the initial PCR-generated clone. The triangle indicates an insertion found in clone RPRl3 relative to clone RPRl 1. B, DNA sequence and predicted amino acid sequence of the RPRC cDNA clone. C, Hydropathy plot of the rat GHRH receptor protein. Seven strongly hydrophobic regions large enough to span the plasma membrane are numbered l7. The GenBank accession number for the RPRC cDNA clone is LO1407.

ments, which are shown in Fig. 5C. The E& for competition of radioligand binding by hGHRH was found to be approximately 50 PM. These values are in reasonable agreement with the reported dissociation constants for binding of hGHRH (l-44)-amide or the

related agonist [His’,Nle”]hGHRH (1-32)-amide to rat pituitary membranes, which range from 41-680 PM (19, 20, 42, 43). The binding specificity was examined in competition experiments, which are shown in Fig. 5D. No specific binding to control 293-WT cells was ob-

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MOL 1738

ENDO.

Vo16No.10

RRMWG-AHVFCV-LSPLPTVLGHM-------HPECDFITQLRE-DE SLLWA-TWVLCL-LNLWGVALGHL-------HLECDFITQLRD-D STMRPRLSLLLLRLLLLTKAAHTVGVPPRLCDVRRVLLEE----PPSPPHVRWLCVLAGALACALRPAGSQAASPQHECEYL-QLIEIQ

hGRF-R rGRF-R r&c-R rVIP-R

hGRF-R rGRF-R rSec-R rVIP-R

GALGPETA -----ETI3

hGRF-R rGRF-R rSec-R rVIP-R

158 158 171 172

V--PLELLAEEES--YFS V--PLELLTEEKS--YFS VNINNSFNERRHA--YL LNDRASSLDEQQQTKFY

hGRF-R rGRF-R rSec-R rVIP-R

218 218 231 232

hGRF-R rGRF-R rSec-R rVIP-R

278 278 291 292

hGRF-R rGRF-R rSec-R rVIP-R

SP AS ss

hGRF-R rGRF-R rSec-R rVIP-R

hGRF-R rGRF-R rSec-R rVIP-R

A-

338 338 351 352

PAQGSLHTQS PAQGGLHT TQETRGSE PPDIGKND

PEL PEL LQE LQG

SA DNF

LpAmTm--W-------------m

LPARRTCTE--W-------------FPLRPVAFNNSF--SNATNGPTHS-VLGWSSKSQHPWGGSNGATCSTQVS

PSRSAAKVLJ---PPRSRVKVL---STEQSRSI---

398 398

410 412 423 423

C C I V

449 459

Fig. 3. Comparison of the Human and Rat GHRH Receptor and Rat Secretin and VIP Receptor Proteins Amino acid residues identical in all four receptors are boxed (28% overall identity). Heavy lines indicate hydrophobic membranespanning domains 1-7, which are placed based on an alignment of the VIP, secretin, CT, and PTH receptors (41). Dots indicate six cysteine residues conserved in all four receptors. The arrowhead points to a site for potential N-linked glycosylation of the GHRH receptors. The carat is over the site at which the 41-amino acid insertion in RPRl3 is found, following tyrosine 324 of the rat GHRH receptor.

served, and the binding to 293-HPR9 cells could be completely competed to 293-WT levels by the addition of excess unlabeled hGHRH (l-44)-amide (Fig. 5D) or the commonly used GHRH agonist [His’,Nlez7]hGHRH (l-32)-amide (not shown). Secretin did not compete binding of the GHRH radioligand at all, and VIP competed binding of the GHRH radioligand only weakly (E&, >l PM). These results demonstrate that the protein encoded by HPR3 is a high affinity GHRH receptor. To determine whether GHRH binding to 293 cells transfected with the cloned GHRH receptor results in

the activation of adenylate cyclase, cells were stimulated with GHRH, secretin, or VIP, and intracellular CAMP levels were measured by RIA. Increasing doses of GHRH resulted in elevated CAMP levels in the 293HPR9 cells (Fig. 6A). The Er& for CAMP accumulation was approximately 5 nM, which is substantially higher than the E& determined in binding-competition experiments. There are several possible explanations for this, including desensitization of the receptor in the presence of isobutylmethylxanthine and high ligand concentrations,

inefficient

coupling

of the

over-expressed

recep-

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Pituitary GHRH Receptor cDNAs

1739

Fig. 4. Production of a Variant Form of the Rat GHRH Receptor by Alternative RNA Processing A, DNA sequence and predicted amino acid sequence of the 123-basepair insertion found in cDNA clone RPR13. B, Model for the generation of cDNA clones RPRll and RPR13 via alternative RNA processing. The upper splicing pattern would generate the RPRll cDNA sequence, while the lower splicing pattern would generate the RPR13 cDNA sequence. Exon sequences are capitalized and underlined and include the predicted amino acid sequence. lntron sequences are in lower-case letters. This model predicts the recognition of a nonconsensus sequence (gc rather than gt, shown in bold) at the 5’-end of the intron following the alternative exon.

tor to downstream components of the signal transduction system in this cell line, and degradation of the ligand by cell- or serum-associated proteases in the absence of protease inhibitors, which were present in the in vitro binding experiments (26). The specificity of GHRH action on these transfected cells is demonstrated in Fig. 6B. GHRH treatment resulted in a large increase in CAMP levels in 293-HPR9 cells but had very little effect on CAMP levels in the control 293-WT cells. Secretin had no effect on CAMP levels in the transfected 293-HPR9 cells, but VIP did increase CAMP to about 5% of the level obtained with GHRH (Fig. 6B), consistent with the ability of VIP to bind to the receptor with low affinity. Expression

of GHRH Receptor

mRNA in Pituitary

The tissue distribution of GHRH receptor mRNA expression was examined in the rat using the RPR64 cDNA as a hybridization probe. Analysis of RNA from several endocrine and nonendocrine tissues is shown in Fig. 7A. A predominant transcript of approximately 2.5 kilobases, as well as a less abundant transcript of approximately 4 kilobases, are observed specifically in the pituitary lane. Figure 7B shows that all lanes of the RNA blot contained roughly equivalent amounts of RNA. The pituitary expression of the rat GHRH receptor was also analyzed by in situ hybridization using 35Slabeled RNA probes. As shown in Fig. 7C, GHRH mRNA was not detected in the liver but was detected in the pituitary using an antisense RNA probe. No hybridization was observed when a control sense-strand GHRH receptor probe was used. The GHRH receptor mRNA colocalized with the mRNA for the pituitary-specific transcription factor Pit-l (44, 45) and was completely restricted to the anterior lobe of the pituitary gland (Fig. 7C).

None Concentration

01 hGRF(l-44,NH2

(M)

GRF Competing

None

GRFSeaelnYlP PeptIde

Fig. 5. Binding of GHRH by the Human GHRH Receptor cDNA Clone HPR3 in Transfected Human Kidney 293 Cells A, Binding of [‘251-Tyr’o]hGHRH (1-44)-amide to membranes from 293-HPR9 cells. The symbols are 0, total binding; A, specific binding; and 0, nonspecific binding, which was determined by inclusion of excess unlabeled GHRH (1 PM) in parallel samples. Values shown are the means of duplicate samples, which varied by less than 5%. B, Scatchard analysis of the binding data. Regression analysis was performed using Data Desk 2.0 software. A dissociation constant of 27 PM and 34 PM was determined in two independent experiments, one of which is shown here. C, Binding competition curve for GHRH in 293-HPR9 cells. The results of two experiments are shown (0 and 0); each was performed in triplicate, and the mean values are shown (values varied by less than 5%). In Exp 1, each tube contained 55 fmol [‘“51-Tyr’o]hGHRH (l-44)-amide, the total bound in the absence of competitor was 25 fmol, and the nonspecific binding was 6.3 fmol. In Exp 2, each tube contained 56 fmol [‘z51-Tyr’o]hGHRH (1-44)-amide, the total bound in the absence of competitor was 16.5 fmol, and the nonspecific binding was 5.9 fmol. The ECsO for competition was approximately 50 PM. D, Specificity of binding of GHRH to 293-HPR9 cells. The amount of input radioligand bound is shown as a function of the cell type (293-WT or 293-HPR9) and the competing peptide, which was present at 1 PM. The dotted line indicates nonspecific binding. Measurements were made in quadruplicate and varied by less than 5%. Each tube contained 42 fmol [1z51-Tyr’0]hGHRH (l-44)-amide, the total bound in the absence of competitor was 14.5 fmol, and nonspecific binding was 3.6 fmol.

DISCUSSION

The structures of the human GHRH receptor, and of a probable rat homolog, have been determined by analysis of pituitary cDNA clones. Evidence that the human cDNA, HPR3, encodes a GHRH receptor includes the following: 1) the encoded protein has the features expected of a G protein-coupled receptor; 2) the encoded protein is highly homologous to the receptors for secretin and VIP, ligands that are in turn related to GHRH; 3)

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MOL 1740

ENDO.

1992

Vo16No.10

0 10-1’

1 o-‘0 Concentration

10-Q 10-Q of hGRF(1-44)NH2

1 o-7 (M)

10-0 Peptids

Hormone

Fig. 6.

Stimulation of CAMP Production by GHRH in Human Kidney 293 Cells Expressing the Human GHRH Receptor A, Stimulation of CAMP production by GHRH in 293-HPR9 cells. The results from two independent experiments are shown (0 and 0); each was performed in triplicate, and the symbols represent the mean r SE. In Exp 1, CAMP levels in the absence of hormone were 4.5 pg/plate of cells, and CAMP levels in the presence of 1 hi hGHRH were 1182 pg/plate of cells. In Exp 2, CAMP levels in the absence of hormone were 6.6 pg/plate of cells, and CAMP levels in the presence of 1 PM hGHRH were 1098 pg/plate of cells. The assay blank for a corresponding vol of buffer was 0.3 pg. B, Specificity of CAMP production by 293-HPR9 cells. Relative CAMP levels are shown as a function of the cell type (293-WT or 293-HPR9) and the inducing peptide, which was present at 1 PM. The dotted line shows basal CAMP levels. All measurements were performed in triplicate and varied by less than 5%, with the exception of the GRF induction of 293-HPR9, where values deviated from the mean by 15%. Actual CAMP amounts are those indicated for Exp 1 above.

Liver 428S-+

Pit-S

Pit-AS

Fig. 7. Expression of GHRH Receptor mRNA in Rat Tissues A, RNA blot analysis of 10 pg polyadenylated RNA from the indicated tissues probed with cDNA clone RPR64. Two transcripts of approximately 2.5 and 4 kilobases are observed only in the pituitary lane. The arrows indicate mobility of the ribosomal RNAs. B, The same blot was subsequently probed with clone CHO-B to ensure that all lanes contained RNA. Autoradiographic exposure in A is for 24 h, and in B is for 6 h. C, In situ detection of GHRH receptor mRNA in the rat pituitary. Adjacent tissue sections probed with RPR64, or with a rat Pit-l cDNA isolated in this laboratory, are shown. Tissues shown are liver and pituitary; pituitary sections were hybridized to both sense and antisense-strand RNA probes.

expression of the cDNA confers specific GHRH binding capacity to transfected human kidney 293 ceils; and 4) expression of the cDNA results in the GHRH-stimulated production of CAMP, the known second messenger for GHRH action, in transfected human kidney 293 cells. While the rat cDNA has not been subjected to these same functional tests, it does encode a protein that is highly homologous to its human counterpart (82%

amino acid identity), and the rat mRNA is specifically expressed in the pituitary gland, the major target for GHRH action. It will be important to test the binding and functional characteristics of the RPRC receptor protein using the homologous rat GHRH as the ligand. The human and rat receptors described here are related to the recently isolated secretin and VIP receptors (40, 41). While most of the homology is contained

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Pituitary GHRH Receptor cDNAs

within the potential membrane-spanning domains (particularly domains 1,2, 5,6, and 7), substantial homology is also found within the cysteine-rich N-terminal domain. This N-terminal region is likely to represent the extracellular ligand-binding domain, and it contains six cysteines that are conserved in the GHRH receptors as well as the secretin and VIP receptors (Fig. 3). This suggests that these cysteines are important for appropriate folding of the extracellular domain into a general structure able to recognize peptides in the secretin, VIP, and GHRH family. Potential N-linked glycosylation sites are not as well conserved between the receptors; there is a conserved glycosylation motif at amino acid 50 of the human and rat GHRH receptors that is not found within the secretin and VIP receptors, although the secretin and VIP receptors do contain other Nlinked glycosylation motifs. The secretin and VIP receptors are more highly related to each other (45% identity) than either is to the GHRH receptor (35% and 40% identity), and all three of these receptors that recognize related ligands are similar to the CT and PTH receptors (38, 39) which recognize structurally distinct ligands. Interestingly, unlike several other genes encoding G protein-coupled receptors, which lack introns in their coding sequence (7, 46), the rat GHRH receptor gene is quite large and is interrupted by numerous introns within the coding region (Rahal, J., E. Zwergel, and K. E. Mayo, unpublished results). The identification of a rat cDNA clone (RPRIS) that contains an in-frame insertion at a point identified as an intron-exon boundary in the gene (Fig. 4) suggests that alternative RNA processing might be used to generate isoforms of the GHRH receptor. The insertion found in clone RPR13 alters the predicted third cytoplasmic loop of the receptor, a region in the fl-adrenergic receptor that is thought to interact with Gs (47,48). Alternative RNA processing also generates two isoforms of the D2 dopamine receptor that differ by an insertion of 29 amino acids into the predicted third cytoplasmic loop (49, 50). In this case, the longer form appears to be predominant in most tissues, and the two isoforms of the receptor have very similar ligand-binding properties (49, 50). It will be important to determine if this alternate GHRH receptor transcript is produced at significant levels in the pituitary, if its expression is hormonally or developmentally regulated, and if the two receptor forms differ in their ability to bind ligand or interact with intracellular effector systems. Because the human GHRH receptor cDNA clone was isolated from a pituitary tumor library, it is possible that it represents an altered form of the receptor. However, functional studies with the human GHRH receptor expressed in human kidney 293 cells reveal that this protein binds GHRH with extremely high affinity, and that this leads to a ligand-dependent increase in the production of intracellular CAMP. These experiments on 293 cells transfected with the receptor cDNA are consistent with earlier studies performed with pituitary cells that demonstrated specific GHRH binding sites. Using the analog [His’,lz5 I-Tyr’“,Nlez7]hGHRH (1-32)-

1741

amide, Seifert et al. (20) originally measured a dissociation constant in competition experiments of 41 PM, in excellent agreement with the 30-PM value measured here in saturation studies or the 50-PM EC0 value determined in competition experiments using the ligand [‘251-Tyr’o]hGHRH (l-44)-amide (Fig. 5). These experiments on 293 cells transfected with the receptor cDNA are also consistent with earlier experiments performed with pituitary cells that suggested that CAMP is a predominant second messenger for GHRH action (24, 25) in that GHRH leads to more than a 1 OO-fold increase in intracellular CAMP in the 293-HPR9 cells (Fig. 6). In addition to its actions on the CAMP pathway, GHRH has been reported to stimulate calcium mobilization and phosphotidylinositol turnover (51, 52) indicating that a secondary pathway using these second messengers might also be operative. It should be possible to more fully examine GHRH-dependent signaling mechanisms using cell lines that express these cloned GHRH receptor cDNAs. It is possible that additional GHRH receptors exist. Two related receptors for the hypothalamic neuropeptide somatostatin, which suppresses GH secretion, have recently been described (7). These two proteins have very similar pharmacological properties for somatostatin binding yet are only 46% identical and vary substantially in their tissue-specific patterns of expression. GHRH mRNA or peptide are reported to be produced in several peripheral tissues, including the placenta and gonads (53-55), and specific GHRH binding to ovarian granulosa cells has been described (56, 57). Using the rat GHRH cDNA described here as a probe, an mRNA transcript was detected only in the pituitary gland, and this mRNA was further localized to the anterior pituitary using in situ hybridization (Fig. 7). Because this mRNA was not detected in the ovary, additional GHRH receptor forms might exist in this or other nonpituitary tissues. Alternatively, the GHRH receptor mRNA could be expressed in the ovary at very low levels, and hence not be easily detected by RNA blot analysis. Last, GHRH might interact with an ovarian VIP receptor (56-58). It is also possible that the receptors characterized here will have affinity for additional peptides in the VIP, secretin, glucagon, GIP, PHI, and PACAP family. The recently characterized VIP receptor binds PACAP with high affinity and binds secretin with much lower affinity (41). The protein encoded by the human cDNA, HPR3, shows specificity for GHRH compared to secretin and VIP in both binding assays and in functional CAMP accumulation assays. Other peptides in this family have not yet been examined for their interaction with the cloned human GHRH receptor. However, pharmacological binding studies suggest that peptides such as GIP and PACAP have unique high affinity receptors, that these receptors do not appreciably bind related peptides such as GHRH, and that these receptors have a distinct tissue distribution from that reported here for the GHRH receptor (59, 60). Defects in GHRH-dependent signal transduction

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MOL 1742

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Vo16No.10

could play a role in some growth disorders, particularly those in which somatotropes capable of GH synthesis are present but GH levels are low (61, 62). The little mouse is a model for this class of disorders; somatotropes from this animal are completely unresponsive to GHRH but release GH in response to CAMP, forskolin, or cholera toxin (34). Alterations in GHRH-dependent signal transduction are also likely to play a role in the development of GH-secreting pituitary adenomas, since the activity and proliferation of the somatotrope cells are controlled in part by GHRH (27-30, 63). Mutations that constitutively activate Gals by inhibiting its intrinsic GTPase function are found in one class of these tumors (31,32), and it seems likely that mutations in the GHRH receptor that activate the receptor in the absence of ligand would result in increased somatotrope proliferation and excess GH secretion. Last, GH secretion becomes impaired during aging, and this is correlated with a decrease in GHRH-stimulated adenylate cyclase activity and a decrease in high affinity GHRH binding sites (42, 64). The isolation of GHRH receptor cDNA probes provides an opportunity to examine directly the involvement of the GHRH receptor in these types of disorders of GH secretion.

MATERIALS Isolation

AND METHODS

and Analysis

of GHRH

Receptor

Clones

Degenerate oligonucleotide primers to membrane spanning domains 6 (5’-ACCCTC[CGA]TNCTG~GA~T~CG~CCGCTC~TC~ T[Tc]GG-3’) .anCi 7 (5’-iGCAC[CT]TCA[cT][Cc][AG]TTG[‘ACj ~AG~~AG~AA~AG~CA~AG~TA-~‘) of the secretin. CT. and PTH ;eceptois were used io amplify cDNA generated from male rat pituitaries. Amplification was carried out for 30 cycles using an annealing temperature of 55 C on a Perkin Elmer Cetus thermocycler and Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, CT). The product was gel-purified and cloned into pGEM3Z (Promega, Madison, WI) for DNA sequence analysis. One of the clones isolated, RPRG, was subsequently used to screen human and rat cDNA libraries, which were from Clonetech (Palo Alto, CA). The human pituitary library was constructed from a GH-producing adenoma from a female patient in X-bluemid; the rat pituitary library was constructed from adult males in X-gtl 0. Screening and plaque purification were performed using standard methods, and inserts were subcloned into pGEM3Z or pGEM7Z (Promega) for further analysis. cDNA clones were sequenced on both strands using modified T7 polymerase (United States Biochemicals, Cleveland OH) and dideoxynucleotides. DNA sequence manipulations were performed using GeneWorks 2.1 software from lntelligenetics (Mountain View, CA). Hydrophobicity was analyzed using the algorithm of Kyte and Doolittle (65) with an analysis window of 11 amino acids. Amino acid sequence alignment was performed using GeneWorks 2.1 software and was subsequently manually modified to maximize alignment of the cysteine residues. Cell Transfection, Measurements

Binding

Assays,

and CAMP

Human cDNA clone HPR3 was subcloned into the eukaryotic expression vector pcDNA-1 (Invitrogen, San Diego, CA) and was used to transfect human kidney 293 cells (a gift from Dr. Barbara Wu, Northwestern University). Transfections were

performed using lipofectin reagent (GIBCO-BRL, Gaithersburg, MD), and 10 fig DNA/l 00-mm plate at a 9:l ratio of the HPR3 cDNA expression construct to pSV2neo (66). Transfected cells were selected in 400 fig/ml G418 (Sigma Chemical, St. Louis, Ml), and individual clones were isolated and expanded for subsequent analysis. Clone HPR9 was selected for further analysis based on high-level expression of the cDNA and was maintained continu&sly in 460 pg/ml G418. For binding assays, control 293-WT cells or transfected 293-HPR9 cells were washed with PBS and homogenized (20 strokes in a Teflon-glass unit) on ice in 50 mM T&-HCI, PH 7.4, 5 mM MgCI*, 2 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged for 5 min at 100 x g to remove larger material, and the supernatant was recentrifuged for 10 min at 4000 x g. The membrane pellet was resuspended in binding buffer: 25 mM HEPES, pH 7.4, 50 mM NaCI, 5 mM MgCI,, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml leupeptin, 1 mg/ml bacitracin, and 0.1% BSA. Binding reactions were performed at 23 C for 60 min in a vol of 0.5 ml with approximaiely 50 pg membrane protein. For competition experiments. 11z51-Tvr’olhGHRH (l-44)-amide (Amersham. Arlington Heights, IL) ‘was present‘at 7i-100 ph. Binding reactions were terminated by centrifugation in a microfuge for 5 min in the cold. The supernatant was aspirated, and the tip of the tube containing the pellet was cut off and counted in a ycounter. Total binding was 30-45% of input, while nonspecific binding was 8-l 1% of input. For CAMP determinations, cells were grown in six-well plates and were treated with 0.1 mM isobutylmethylxanthine for 20 min at 37 C. Hormones were added in fresh warmed media, and the incubation was continued for another 20 min at 37 C. Medium was removed, 0.3 ml cold 0.1 N HCI was added to each well, and cell lysates were stored at -20 C until CAMP RlAs were performed as described (67). All peptides were the human sequences, and they were obtained from Peptides International (Louisville, KY) or Peninsula Laboratories (Belmont, CA). RNA Blot Analysis

and in Situ Hybridization

RNA was prepared from the indicated tissues by homogenization in guanidine isothiocyanate and centrifugation through cesium chloride, and polyadenylated RNA was selected by chromatography using oligo(dT) cellulose. Approximately 10 fig of each RNA were separated by electrophoresis on denaturing 1% agarose/formaldehyde gels. RNA was transferred to a nylon membrane (ICN, Irvine, CA), covalently attached by UV cross-linking, and detected by hybridization to the insert from RPR-64 that had been labeled with [32P]dCTP using random hexamer primers and the Klenow fragment of Escherichia co/i DNA polymerase. Hybridization was performed in 50% formamide, 5x SSPE (1 x SSPE = 0.15 M NaCI, 0.01 M NaH,P04, 1 mM EDTA, pH 7.4), 2x Denhardt’s reagent, 10% dextran sulfate, 0.1% sodium dodecyl sulfate, and 100 pg/ml yeast tRNA. The membranes were subsequently washed in 6.1~ SSC (lx SSC = 0.15 M NaCI, O.Ol$ M Na citrate, pH 7.0) at 65 C and exposed to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY). After removal of probe in 50% formamide at 65 C, the membranes were rehybidized to cDNA clone CHO-B (681. which detects the LLReo3 aene familv (69). to assess the‘a&unt of RNA present in each-lane. ’ ~ ” For in situ hybridization, frozen tissues were removed from storage at -80 C and brought to -20 C, and 20-Frn sections were cut using a Reichert 820 cryostat (Buffalo, NY). Sections were mounted onto gelatin- and poly L-lysine-coated glass slides for processing as described previously (70). In brief, liver or pituitary sections were fixed in 5% paraformaldehyde (pH 7.5) for 5 min, washed in 2x SSC followed by 0.1 M triethanolamine (pH 8.0) and incubated in 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min. Sections were dehydrated using ethanol and vacuum dried. Antisense and sense [35S]UTP-labeled RNA probes were synthesized using T7 or SP6 polymerase. The RNA probe (2 x lo7 cpm/ml in

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Pituitary

GHRH

Receptor

cDNAs

hybridization buffer: 50% formamide, 5x SSPE, 2x Denhardt’s reagent, 10% dextran sulfate, 0.1% sodium dodecyl sulfate, and 100 pg/ml yeast tRNA) was applied, and the tissue sections were overlaid with a coverslip. Slides were hybridized in a humidity chamber at 47 C for 16-l 8 h. After hybridization, the coverslips were removed, and sections were treated with RNase A (20 ualml) at 37 C for 30 min. washed in increasinalv lower concentrations of SSC down to 0.1 x SSC at 55 C, a&f dehydrated using ethanol. The slides were exposed to betamax film (Amersham) for 2-3 days and were then processed for liquid emulsion autoradiography using NTB-2 emulsion (Eastman Kodak Co.).

1743

12. 13. 14. 15.

Acknowledgments

16.

I thank members of my laboratory for many insightful discussions and for their patience during the course of this work, Jason Rahal and Eberhard Zwergel for help with cDNA sequencing, Joe Takahashi for CAMP measurements, and Dan Linzer, Barbara Wu, Joe Takahashi, and Rick Gaber for comments on the manuscript.

17.

Received July 30, 1992. Revision received August 27, 1992. Accepted August 27, 1992. Address requests for reprints to: Dr. Kelly E. Mayo, Department of Biochemistry, Molecular Biology, and Cell Biology, 2153 Sheridan Road, Hogan Science Building, Northwestern University, Evanston, Illinois 60208. This work was supported by a NIH Research Career Development Award (to K.E.M.).

18.

19.

20.

21.

22. REFERENCES 23. 1. Martin JB 1979 Brain mechanisms for integration of growth hormone secretion. Physiologist 22:23-29 2. Hall K, Sara VR 1983 Growth and somatomedins. Vitam Horm 40: 175-233 3. Muller EE, Cocchi D, Locatelli V (eds) 1989 Advances in Growth Hormone and Growth Factor Research. Springer Verlag, Berlin 4. Ullrich A, Gray A, Tam A, Yang-Feng T, Tsubokawa M, Collins C, Henzel W, LeRon T, Kathuria S, Chen E, Jacobs S, Francke U, Ramachandran J, Fujita-Yamaguchi Y 1986 Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J 5:2503-2512 5. Morgan D, Edman J, Standring D, Fried V, Smith M, Roth R, Rutter W 1987 Insulin-like growth factor II receptor as a multifunctional binding protein. Nature 329:301-307 6. Leung D, Spencer S, Cachianes G, Hammonds R, Collins C, Henzel W, Barnard R, Waters M, Wood W 1987 Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330537-543 7. Yamada Y, Post S, Wang K, Tager H, Bell G, Seino S 1992 Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney. Proc Natl Acad Sci USA 89:251-255 8 Czech MP 1989 Signal transmission by the insulin-like growth factors. Cell 59:235-238 9 Humbel RE 1990 Insulin-like growth factors I and II. Eur J Biochem 190:445-462 10 Kelly PA, Djiane J, Postel-Vinay M-C, Edery M 1991 The prolactin/growth hormone receptor family. Endocr Rev 3:235-251 11 Godowski PJ, Leung DW, Meacham LR, Galgani JP, Hellmis R, Keret R, Rotwein PS, Parks JS, Laron Z, Wood WI 1989 Characterization of the human growth hormone

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

receptor gene and demonstration of a partial gene deletion in two patients with Laron-type dwarfism. Proc Natl Acad Sci USA 86:8083-8087 Ross EM 1989 Signal sorting and amplification through G protein-coupled receptors. Neuron 3:141-l 52 Frohman L,.Jansson J-O 1986 Growth hormone-releasing hormone. Endocr Rev 7:223-253 Gelato MC, Merriam CR 1986 Growth hormone releasing hormone. Annu Rev Physiol48:569-591 Guillemin R, Brazeau P, Bohlen P, Esch F, Ling N, Wehrenberg WB 1982 Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science 218:585-587 Rivier J, Spiess J, Thorner M, Vale W 1982 Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nature 300:276-278 Bell GI 1986 The glucagon superfamily: precursor structure and gene organization. Peptides 7 (Suppl):27-36 Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Jiang L, Culler MD, Coy DH 1989 Isolation of a novel 38 residue hypothalamic polypetide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 164:567-574 Abribat T, Boulanger L, Gaudreau G 1990 Characterization of [‘251-Tyr”‘]human growth hormone-releasing factor (l-44) amide bindinq to rat pituitary: evidence for high and low affinity classes of sites. Brain Res 528:291-299 Seifert H. Perrin M. Rivier J. Vale W 1985 Bindina sites for growth hormone releasing factor on rat anterior pituitary cells. Nature 313:487-489 Velicelebi G, Santacroce T, Harpold M 1985 Specific binding of synthetic human pancreatic growth hormone releasing factor (l-40-OH) to bovine anterior pituitaries. Biochem Biophys Res Commun 126:33-39 Velicelebi G, Patthi S, Provow S, Akong M 1986 Covalent cross-linking of growth hormone-releasing factor to pituitary receptors. Endocrinology 118:1278-l 283 Zysk J, Cronin M, Anderson J, Thorner M 1986 Crosslinking of a growth hormone releasing-factor binding protein in anterior pituitary cells. J Biol Chem 261:1678116784 Bilezikjian L, Vale W 1983 Stimulation of adenosine 3’,5’monophosphate production by growth hormone-releasing factor and its inhibition by somatostatin in anterior pituitary cells in vitro. Endocrinology 113:1726-l 731 Labrie F, Gagne 8, Lefevre G 1983 Growth hormonereleasinq factor stimulates adenvlate cvclase activity in the anterior pituitary gland. Life Sci 33:2229-2233 Struthers R. Perrin M. Vale W 1989 Nucleotide reaulation of growth hormone-releasing factor binding to rat pituitary receptors. Endocrinology 124:24-29 Barinaga M, Yamamoto G, Rivier C, Vale W, Evans R, Rosenfeld MG 1983 Transcriptional regulation of growth hormone gene expression by growth hormone-releasing factor. Nature 306:84-85 Gick GG, Zeytin F, Brazeau P, Ling NC, Esch F, Bancroft FC 1984 Growth hormone-releasing factor regulates growth hormone mRNA in primary cultures of rat pituitary cells. Proc Natl Acad Sci USA 81 :1553-l 555 Billestrup N, Mitchell R, Vale W, Verma I 1987 Growth hormone-releasing factor induces c-fos expression in cultured primary pituitary cells. Mol Endocrinol 1:300-305 Billestrup N, Swanson LW, Vale W 1986 Growth hormone-releasing factor stimulates proliferation of somatotroohs in vitro. Proc Natl Acad Sci USA 83:6854-6857 Landis C, Masters S, Spada A, Pace A, Bourne H, Vallar L 1989 GTPase inhibiting mutations activate the a chain of Gs and stimulate adenyl cyclase in human pituitary tumours. Nature 340:692-696 Vallar L, Spada A, Giannattasio G 1987 Altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas. Nature 330:566-568 Cooper JA 1990 Oncogenes and anti-oncogenes. Curr Opin Cell Biol 2:285-295

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MOL 1744

END0.1992

34. Jansson J-O, Downs T, Beamer W, Frohman L 1986 Receptor-associated resistance to growth hormone-releasing factor in dwarf “little” mice. Science 23251 l-51 2 L 1991 Evidence for a defect in growth 35. Downs T, Frohman hormone-releasing factor signal transduction in the dwarf (dw/dw) rat pituitary. Endocrinology 129:58-67 J 1979 Inherited ateliotic dwarfism in 36. Either E, Beamer mice: characteristics of the mutation, little, on chromosome 6. J Hered 67:87-91 37. Libert F, Parmentier M, Lefort A, Dinsart C, Van Sande J, Maenhaut C, Simons M-J, Dumont JE, Vassar-t G 1989 Selective amplification and cloning of four new members of the G protein-coupled receptor family. Science 244:569-572 38. Lin H, Tonja H, Flannery M, Aruffo A, Kaji E, Gorn A, Kolakowski LJ, Lodish HF, Goldring S 1991 Expression cloning of an adenylate cyclase-coupled calcitonin receptor. Science 254:1022-l 024 39. Juppner H, Abdul-Badi A-S, Freeman M, Kong X, Schipani E, Richards J, Kolakowski LJ, Hock J, Potts JT, Kronenberg H, Segre G 1991 A G-protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide. Science 254:1024-l 026 40. lshihara T, Nakamura S, Toro Y, Takahashi T, Takahashi K, Nagata S 1991 Molecular cloning and expression of a cDNA encoding the secretin receptor. EMBO J 10:16351641 41. lshihara T, Shigemoto R, Mori K, Takahashi K, Nagata S 1992 Functional expression and tissue distribution of a novel receptor for vasoactive intestinal peptide. Neuron 8:811-819 42. Abribat T, Deslauries N, Brazeau P, Gaudreau P 1991 Alterations of pituitary growth hormone-releasing factor binding sites in aging rats. Endocrinology 128:633-635 43. Seifert H, Perrin M, Rivier J, Vale W 1985 Growth hormone-releasing factor binding sites in rat anterior pituitary membrane homogenates: modulation by glucocorticoids. Endocrinology 117:424-426 44. Bodner M, Castrillo J-L, Theill L, Deerinck T, Ellisman M, Karin M 1988 The pituitary-specific transcription factor GHF-1 is a homeobox-containing protein. Ceil 55:505518 45. lngraham H, Chen R, Mangalam H, Elsholtz H, Flynn S, Lin C, Simmons D, Swanson L, Rosenfeld M 1988 A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 55:519-529 46. O’Dowd B, Lefkowitz R, Caron M 1989 Structure of the adrenergic and related receptors. Annu Rev Neurosci 12:67-83 47. Strader C, Sigal I, Dixon R 1989 Structural basis of padrenergic receptor function. FASEB J 3:1825-l 832 48. O’Dowd R, Hrantowich M, Regan J, Leader W, Caron M, Lefkowitz R 1988 Site-directed mutagenesis of the cytoplasmic domain of the human P2-adrenergic receptor. J Biol Chem 263:15985-l 5992 49. Monsma F, McVittie L, Gerfen C, Mahan L, Sibley D 1989 Multiple D2 dopamine receptors produced by alternative RNA splicing. Nature 342:926-929 50. Giros B, Sokoloff P, Martres M-P, Riou J-F, Emorine L, Schwartz J-C 1989 Alternative splicing directs the expression of two D2 dopamine receptor isoforms. Nature 342:923-926 51. Login IS, Judd AM, MacLeod RM 1986 Association of ‘%a’+ mobilization with stimulation of growth hormone (GH) release by GH-releasing factor in dispersed normal male rat pituitary cells. Endocrinology 118:239-243 52. Canonico PL, Cronin MJ, Thorner MO, MacLeod RM 1983 Human pancreatic GRF stimulates phosphotidylinositol labeling in cultured anterior pituitary cells. Am J Physiol 245:E587-E590 53 Bagnato A, Moretti C, Ohnishi J, Frajese G, Catt K 1992 Expression of the growth hormone-releasing hormone

Vo16No.10

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

gene and its peptide product in the rat ovary. Endocrinology 130:1097-l 102 Berry SA, Pescovitz OH 1990 Ontogeny and pituitary regulation of testicular growth hormone-releasing hormone-like messenger RNA. Endocrinology 127:14041411 Margioris AN, Brockman G, Bohler HLC, Grino M, Vamvakopoulos N, Chrousos GP 1990 Expression and localization of growth hormone-releasing hormone messenger ribonucleic acid in the rat placenta: in vitro secretion and regulation of its peptide product. Endocrinology 126:151158 Bagnato A, Moretti C, Frajese G, Catt K 1991 Gonadotropin-induced expression of receptors for growth hormone releasing factor in cultured granulosa cells. Endocrinology 128:2889-2894 Moretti C, Bagnato A, Solan N, Frajese G, Catt K 1990 Receptor-mediated actions of growth hormone releasing factor on granulosa cell differentiation. Endocrinology 127:2117-2126 Spicer L, Langhout D, Alpizar E, Williams S, Campbell R, Mowles T, Enright W 1992 Effects of growth hormonereleasing factor and vasoactive intestinal peptide on proliferation and steroidogenesis of bovine granulosa cells. Mol Cell Endocrinol 83:73-78 Maletti M, Portha B, Carlquist M, Kergoat M, Laburthe M, Marie JC, Rosselin G 1984 Evidence for and characterization of specific high affinity binding sites for the gastric inhibitory polypeptide in pancreatic p-cells. Endocrinology 115:1324-l 331 Schafer H, Schwarzhohh R, Creutzfeld W, Schmidt WE 1991 Characterization of a guanosine-nucleotide-bindingprotein-coupled receptor for pituitary adenylate-cyclaseactivating polypeptide on plasma membranes from rat brain. Eur J Biochem 202:951-958 Underwood L, Van Wyk JJ 1985 Normal and aberrant growth. In: Wilson JD, Foster DW (eds) Williams’ Textbook of Endocrinology. Saunders, Philadelphia, pp 155205 MacGillivray MH 1987 Disorders of growth and development. In: Felig F, Baxter JD, Broadus AE, Frohman LA (eds) Endocrinoloqv and Metabolism. McGraw-Hill, New York, pp 1581-l 628 Mayo KE, Hammer RE, Swanson LW, Brinster RL, Rosenfeld MG, Evans RM 1988 Dramatic pituitary hyperplasia in transgenic mice expressing a human growth hormone-releasina factor aene. Mol Endocrinol 2:606-612 Robberecht P,-Gillard M, Waelbroek M, Camus J, DeNeef P, Christophe J 1986 Decreased stimulation of adenylate cyclase by growth hormone-releasing factor in the anterior pituitary of old rats. Neuroendocrinology 44:429-432 Kyte J, Doolittle R 1982 A simple model for displaying the hydropathic character of a protein. J Mol Biol 157:105132 Southern P, Berg P 1982 Transformation of mammalian cells to antibiotic resistance with a bacterial gene under the control of the SV40 early region promoter. J Mol Appl Genet 1:327-341 Nikaido S, Takahashi J 1989 Twenty-four hour oscillations of CAMP in chick pineal cells: role of CAMP in the acute and circadian regulation of melatonin production. Neuron 3:609-619 Harpold MM, Evans RM, Salditt-Georgieff M, Darnell JE 1979 Production of mRNA in Chinese hamster cells: relationship of the rate of synthesis to the cytoplasmic concentration of nine specific mRNA sequences. Cell 17:1025-l 035 Heller DL, Gianola KM, Leinwand LA 1988 A highly conserved mouse gene with a propensity to form pseudogenes in mammals. Mol Cell Biol 8:2797-2803 Suhr ST, Rahal JO, Mayo KE 1989 Mouse growth hormone-releasing hormone: precursor structure and expression in brain and placenta. Mol Endocrinol 3:1693-l 700

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