GHS-R Family Members and Ligands/Smith et al. Vol. 14, No. 1 9 Author to whom all correspondence and reprint requests should be addressed: Dr. Roy G. Smith, Huffington Center on Aging, Baylor College of Medicine, M320, One Baylor Plaza, Houston TX 77030-3498. E-mail: rsmith@bcm.tmc.edu Endocrine, vol. 14, no. 1, 9–14, February 2001 0969–711X/01/14:9–14/$11.50 © 2001 by Humana Press Inc. All rights of any nature whatsoever reserved. 9 We have previously reported the cloning and charac- terization of a new orphan G-protein-coupled recep- tor (GPC-R), the growth hormone secretagogue receptor (GHS-R), and shown that this receptor medi- ates the activity of the growth hormone–releasing peptides (GHRPs) and nonpeptide ligands such as L-692,429 and MK-0677. Because the GHS-R obvi- ously does not belong to any of the known GPC-R subfamilies, we searched for GHS-R family members by screening a human genomic library using low-strin- gency hybridization and screening a Pufferfish genomic library. The Pufferfish was selected because of its compact genome. From the human genomic library, a homolog, GPR38, with 52% identity to the GHS-R was isolated. From the Pufferfish library, three family members were isolated. The Pufferfish gene having 58% identity to the GHS-R, on expression in HEK293 cells, was activated with GHRP-6 and MK-0677. These results indicate that the GHS-R has been con- served for at least 400 million years and that the Pufferfish genome is appropriate for isolation of GHS- R family members. In our search for endogenous ligands for the orphan receptors GHS-R and GPR38, we showed that adenosine is a partial agonist of the GHS-R and that motilin is the endogenous ligand for GPR38. We also confirmed that the endogenous ligand ghrelin is a full agonist of the GHS-R. Key Words: Growth hormone secretagogue receptor; motilin; adenosine. Introduction There remain many important issues to be resolved con- cerning the biologic role of the growth hormone secreta- gogue receptors (GHS-Rs) and the identity of endogenous ligands for these receptors. Because the GHS-R appears to be the first of a new family of G-protein-coupled receptors (GPC-Rs) (1), we wished to identify GHS-R family mem- bers and investigate the molecular evolution of the GHS-R. We initially focused on the isolation of GHS-R family members by searching expressed sequence tag (EST) data- bases and screening a human genomic library. It soon became evident that identification of GHS-R family members by the latter approach was an enormous task. We reported the isolation of three GHS-R homologs (GPR38, GPR39, and FM3) from a human genomic PAC library (2,3). However, screening this library provided positive hybridization sig- nals for 325 PAC clones (2). The relatively low percentage of coding sequences in the human genome complicates characterization of GHS-R family members from this num- ber of positive signals. To simplify the process, we evalu- ated the potential of using the Pufferfish, a vertebrate organism with a compact genome. The Pufferfish was selected because it is evolutionarily distant from human and homologous genes are likely conserved because of their functional importance. Our objectives were first to deter- mine whether the orphan GHS-R had been evolutionarily conserved from Pufferfish to humans, and, second, to deter- mine whether this vertebrate species would provide a short- cut for identifying new GHS-R family members. To identify natural ligands for the GHS-R and family members, we developed high-volume screening assays. The assays were developed such that they would be robust enough for iden- tifying compounds in crude tissue extracts. In this review we describe the identification of human GHS-R homologs in the Pufferfish. We also report the iden- tification of adenosine as an endogenous ligand of the GHS- R and motilin (MTL) as the ligand for the closest GHS-R family member, GPR38. Human GHS-R Homolog in Pufferfish Teleost fish have the specialized functions of higher vertebrates; hence, genes necessary for complex functions are present in their genome. The genome of the Japanese Pufferfish (Fugu rubripes) had been proposed by Brenner et al. (4) to be a compact model genome for vertebrates. The Pufferfish genome (400 Mb) has approximately the same Growth Hormone Secretagogue Receptor Family Members and Ligands Roy G. Smith, 1,2 Reid Leonard, 2 Alex R. T. Bailey, 1 Oksana Palyha, 2 Scott Feighner, 2 Carina Tan, 2 Karen Kulju McKee, 2 Sheng-Shung Pong, 2 Pat Griffin, 2 and Andrew Howard 2 1 Huffington Center on Aging and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX; and 2 Merck Research Laboratories, Rahway NJ