Molecular cloning of ovine and bovine growth hormone-releasing hormone receptors: the ovine receptor is C-terminally truncated.
To provide information about species differences in GH-releasing hormone (GHRH) receptors useful for studies of receptor-ligand binding properties and receptor function, we have cloned the ovine and bovine pituitary GHRH receptors (GHRHRs). The ovine receptor (oGHRHR) was cloned from a pituitary complementary DNA library and encodes a protein that is similar to that of porcine, human, rat, and mouse with, respectively, 84.3, 80.7, 75.9, and 74.0% amino acid identity. Surprisingly, oGHRHR has a 16 amino acid truncation at its carboxyl-terminal end when compared with GHRHRs from other known mammals. RT-PCR using pooled pituitary RNA from a different population of sheep could detect only truncated receptor. Bovine GHRHR (bGHRHR) was cloned by RT-PCR and shows 92.5% amino acid sequence identity with oGHRHR, but has no truncation. Genomic sequencing of the appropriate region of goat receptor intron 13 showed that the caprine receptor shares the same truncation seen in sheep. Photoaffinity cross-linking of GHRH to ovine and bovine pituitary membranes confirms that the native ovine pituitary GHRHR protein is smaller by the amount predicted by the cloned sequences. The truncation did not affect GHRH binding as oGHRHR, bGHRHR, human GHRHR, and human GHRHR, which was truncated by site-directed mutagenesis to match the oGHRHR, all showed comparable GHRH binding affinity when expressed in transfected cell lines. In contrast, the ovine and truncated human receptors demonstrated enhanced sensitivity for GHRH stimulation of cAMP (lowered ED(50)) relative to hGHRHR and bGHRHR. This suggests that this C-terminal domain acts to inhibit cAMP signaling possibly through a role in receptor down regulation.
Horikawa
R
R
Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia Health System, Charlottesville, Virginia 22908, USA. rehorikawa@nch.go.jp
Gaylinn
B D
BD
Lyons
C E
CE
Thorner
M O
MO
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle / Q000235:genetics*; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D013007:Growth Hormone-Releasing Hormone / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels; D018013:Receptors, Neuropeptide / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018017:Receptors, Pituitary Hormone-Regulating Hormone / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012756:Sheep / Q000235:genetics*; D015398:Signal Transduction; D013045:Species Specificity
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D019920:Photoaffinity Labels; D018013:Receptors, Neuropeptide; D018017:Receptors, Pituitary Hormone-Regulating Hormone; D013007:Growth Hormone-Releasing Hormone; D000242:Cyclic AMP; C048250:somatotropin releasing hormone receptor