The molecular cloning of a receptor for growth hormone secretagogues (GHSs) from humans and other species provides evidence that a third neuroendocrine pathway exists, in addition to growth hormone releasing hormone and somatostatin, that aids in the control of pulsatile growth hormone (GH) release from the pituitary gland, presumably regulated by an as yet unidentified hormone. Expression cloning was adopted to isolate a cDNA encoding the growth hormone secretagogue receptor (GHS-R; (1)). The isolation of cDNA and genomic clones encoding the GHS-R has been the subject of recent reviews. In brief, earlier investigations aimed at elucidation of the signal transduction pathway activated by GHSs demonstrated that GHSs activate phospholipase C resulting in a rise in inositol triphosphate and intracellular Ca. In contrast, growth hormone releasing hormone (GHRH) signal transduction occurs through the activation of adenylate cyclase and subsequent elevation of intracellular cAMP. ImiidXlyyXenopus oocytes injected with swine pituitary poly (A) mRNA as a source of GHS-R mRNA occasionally gave a modest activation of Ca activated CI currents in response to MK-0677. To improve reproducibihty, assay throughput, and reliability of the response, swine poly (A) mRNA was supplemented with various G subunit mRNAs.
In addition, rather than to measure the activation of phospholipase C by electrophysiological methods, mRNA encoding the Ca sensitive luminescent protein aequorin was co-injected. Using a luminometer, responses were measured generally 2-days postinjection following challenge with MK-0677. MK-0677 responsiveness was dependent on the co-injection of the G family member Gi. Other G subunit tested failed to rescue MK-0677-induced bioluminescence. This observation provided a key biochemical breakthrough in our expression cloning protocol by enhancing assay sensitivity and reproducibility. Once this assay was in place, we injected complex pools of 10,000 cRNAs from unfractionated swine pituitary cDNA libraries and identified a single cDNA which encoded the GHS-R. To date, only a single type of GHS-R has been identified at the molecular level, though additional G-protein coupled receptors (GPC-Rs) that may confer GHS sensitivity have been postulated to exist. The GHS-R identified by expression cloning is a classical GPC-R containing seven putative alpha-helical membrane spanning segments (7-TM) and three intracellular and three extracellular loops. In addition, a highly conserved motif responsible for G protein interaction (D/ERY) found in the second intracellular loop immediately following TM-3 is present in the GHS-R. As noted in other GPC-Rs, consensus sequences for N-linked glycosylation, phosphorylation are present, and cysteines located in the first two extracellular loops capable of disulfide bonding are also found. Molecular analysis of GHS-Rs from swine, human, rat, mouse and dog revealed that the GHS-R is strongly conserved in evolution.
Comparison of the amino acid sequences among these species indicates remarkable overall sequence identity, with only few amino acid substitutions in the TM domains and loop regions. The most divergent GHS-R protein was found in the dog, in which a 17 amino acid segment in the N-terminal extracellular domain are lacking when compared to the other species tested. As both multiple genomic and cDNA clones were isolated, it is unlikely that this is due to a cloning artifact. Despite numerous attempts, other dog GHS-R cDNAs or genomic clones could not be identified suggesting that this clone represents an authentic dog GHS-R. This conclusion is further supported by the pharmacological characterization of the dog GHS-R. The full length human GHS-R gene encodes a 366 amino acid protein, except in rodents where the protein measures 364 residues with a loss of one amino acid in the N-terminal extracellular domain and one amino acid in the second intracellular loop. The rat and mouse forms differ by only two amino acids from each other. From both human and swine pituitary, two types of cDNAs were isolated: type la, encoding a functional protein containing 7-TM domains, and type lb, encoding a protein containing TM-1 through 5 with no measurable functional activity in cell based assays. These two forms most likely arise from transcription of a single gene by alternative mRNA processing.
This assertion was confirmed by determination of the nucleotide sequence for the proposed human exon-intron boundaries and the complete intron of the human gene. As seen in many GPC-Rs, introns are usually placed between TM domains and often following TM-5. Type la cDNA encodes the complete 7-TM GHS-R and results from a splicing event which removes the intron. In type lb cDNA, the intron is not removed and an alternative polyadenylation signal is presumably utilized in the intron. As a result, the human and swine type lb cDNA contain a short, 24-amino acid open reading frame fused to leucine-263 which is conserved in human and swine. Southern blot analysis to search for GHS-R related genes again indicates that the GHS-R is highly conserved since a simple hybridization pattern is observed when high stringency post-hybridizational washing conditions are utilized. Our analysis included numerous mammalian and non-mammalian species, including Drosophila, Ceanorhabditis elegans and teleost fish.
The existence of the GHS-R can apparently be extended to Precambrian times (400,000 million years) as amino acid sequences strongly related to the human GHS-R have been identified in teleost fish (unpublished results). This observation points to the importance of the GHS ligand system in normal physiological function. Evidence for a second GHS-R subtype has been suggested by studies with a photoactive, radiolabeled derivative of hexarelin, a potent and specific growth hormone releasing protein. Covalent crosslinking of this analog to membranes labeled a 57 kDa protein in anterior pituitary and an 85 kDa protein in heart membranes. The protein may be distinct from the GHS-Rla since labeling of the protein was not potently displaced by MK-0677. Molecular cloning is required to unequivocally demonstrate these proteins as potential GHRP receptors.
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