A comprehensive search was initiated for GHS-R related GPC-Rs. Overall, this search was undertaken to further elucidate the mechanism of action of MK-0677 as it proceeds through clinical evaluation in humans. Our rationale for the notion that GHS-R family members the designed oligonucleotide in the target are then recovered after binding to streptavidin beads. In the second phase, we concentrated on the isolation of GHS-R related sequences from lower vertebrates. Interestingly, GHS-R related sequences were indeed detected in the puffer fish Spheroides nephelus, one of the oldest known vertebrates. Several investigators have shown the utility of identifying homologous genes of mammalian origin in puffer fish, most notably from Fugu rubripes.
Puffer fish are estimated to contain -90% of mammauan genes but have a genome size -8-fold smaller than mammals because intron size and repeat sequences are reduced in size and abundance. In studies in progress, three full-length GPC-R genes with good sequence identity (58%, 47% and 34% at the protein level) to the human GHS-R have been isolated and are currently being characterized (unpubUshed results). The search for GHS-R family members first involved identifying a human genomic DNA segment with strong sequence conservation to the human GHS-R. Determination of the nucleotide sequence from this fragment revealed that it partially encodes GHS-R family member 1 (FM-1, GPR38), and served to provide broad access to both new members of the previously unknown GHS-R family as well as other related GPC-Rs. This fragment shares 71% nucleic acid and 67% amino acid sequence identity to the human GHS-R. This hybridization probe was used to successfully isolate clone FM-2 (GPR39), the type 2 galanin receptor, and the GHS-R and type 2 neurotensin-R from rat hypothalamus (unpubHshed results).
Genbank databases were monitored daily using the Tblastn program with amino acid sequence from the human GHS-R TM domains 6-7. Two significant "hits" were detected. A mouse EST derived from a T-cell library was identified with a significant homology score (63% DNA, 36% amino acid sequence identity) to the 3 end of the gene for the human GHS-R. Full length cDNA were then obtained for both the mouse and human forms. The human and murine FM-3 exhibit strong protein sequence identity (73%). In addition, a cosmid clone (K10B4) from the worm C elegans contains an open reading frame encoding a full-length GPC-R with strongest protein sequence identity to the human GHS-R (29%). The open reading frame is contained on five exons. An alignment (Pileup) was generated using the deduced amino acid sequences for GHS-R and related sequences. The intron in GPR38 is positioned between TM 5 and 6 and its location is conserved when compared to the intron in the GHS-R. GPR38 shares 52% identity and 73% similarity with the human GHS-R (73% to 89% similarity in the transmembrane helical domains). GPR39's overall similarity (based on the predicted amino acid sequence of the cDNA) to the type la GHS-R is 52%. All of the GHS-R family members are closely related to the neurotensin receptors (53-58% similarity) with TM2 showing the highest level of conservation (71-91% similarity). Database searching indicates that FM-3 is most closely related to the GHS-R and NT-R type 1 with 33 and 29% protein sequence identity. FM-3 groups as a separate branch distinct from both GHS-R and NT-R sequences and the orphan GPC-Rs GPR38 and GPR39. The apparent rarity of GPR38 transcripts precluded their detection by standard Northern blotting, while GPR39 mRNA could readily be observed by this method.
The more sensitive technique of RNase protection was therefore used to assess the expression pattern of GPR38. RNAse protection with GPR38 revealed expression in human stomach, thyroid and bone marrow. GPR38 transcripts could not be detected in whole brain, hippocampus, pituitary gland, subthalamic nucleus, caudate nucleus, cerebellum, thalamus, placenta, testes, uterus, lung, kidney, or spleen. Other tissues which did not contain detectable GPR38 transcripts included prostate, pancreas, skeletal muscle, thymus, small intestine, adrenal and salivary gland. Unlike the GHS-R where expression in neuroendocrine tissues is consistent with a role in growth hormone function, the significance of GPR38 expression in the thyroid, stomach and bone marrow is yet to be elucidated. Whereas the expression profile of GPR38 was restricted, GPR39 transcripts were detected in many tissues and brain regions. Most, if not all brain regions tested gave a single hybridizing species of 1.8-2 kb. However several peripheral tissues, such as stomach and small intestine contain a transcript, in addition to the -1.8-2 kb band, at - 3 kb which may have arisen by alternative mRNA processing. Interestingly, this 3 kb species is the only hybridizing band noted in other tissues such as pancreas, thyroid and colon. FM-3 was cloned from a mouse T-cell library and its mRNA was readily detected by Northern blot analysis.
The predominate transcript size was 5 kb, which was detected in all tissues examined. A band of 2 kb was also noted in most of the tissues (with high abundance in testis), suggesting alternative mRNA processing of the FM-3 primary transcript. Southern blot analysis (Fig. 3B) of EcoRl-digested genomic DNA using the mouse form as a radiolabeled probe gave a simple hybridization pattern in all species tested, indicative of a single, highly conserved gene encoding FM-3. Attempts to identify activating ligands for any orphan GPCR first involve expression of the full-length contiguous open reading frame for the protein in a suitable heterologous cell type. This can usually be accomplished by placing the ORF in a suitable mammalian expression vector which drives high-level protein expression. It is pivotal to validate that the orphan GPC-R is being expressed appropriately, that is, that a protein of predicted molecular weight is observed and expression at cell membrane is achieved. In the absence of anti-peptide antibodies against the protein, epitope tagging (usually without any negative effect at the N-terminus) the protein can be used to document expression. Then, based on amino acid sequence as a rough guide for its closest relative, functional and ligand binding assays can be utilized to identify a cognate ligand for the orphan GPCR.
Experiments performed to identify the natural ligands for GPR38, 39 and FM-3 have as yet been unsuccessful. Transfected HEK-293 cells expressing each clone separately (cell membrane expression confirmed using epitope-tagged protein) failed to bind radiolabeled MK-0677 or neurotensin. In addition, several peptides including endothelin, VIP, gastrin, growth hormone-releasing hormone, somatostatin, TRH, calcitonin, and galanin did not activate FM-3 as measured by the aequorin bioluminescence assay which senses IP3-induced Ca^^ mobilization as a result of phospholipase C activation (data not shown). The identification of an activating ligand for these novel GPC-Rs remains to be discovered.
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1. The hypothalamus and other neuronal markers
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