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The rainbow trout (Oncorhynchus mykiss) is one of the most widely used fish species in aquaculture and physiological research. In the present paper, we report the first cloning, 3D (three-dimensional) modelling, pharmacological characterization and tissue distribution of two melanocortin (MC) receptors in rainbow trout. Phylogenetic analysis indicates that these receptors are orthologues of the human MC4 and MC5 receptors. We created 3D molecular models of these rainbow trout receptors and their human counterparts. These models suggest greater divergence between the two human receptors than between their rainbow trout counterparts. The pharmacological analyses demonstrated that ACTH (adrenocorticotropic hormone) had surprisingly high affinity for the rainbow trout MC4 and MC5 receptors, whereas alpha-, beta- and gamma-MSH (melanocyte-stimulating hormone) had lower affinity. In second-messenger studies, the cyclic MSH analogues MTII and SHU9119 acted as potent agonist and antagonist respectively at the rainbow trout MC4 receptor, indicating that these ligands are suitable for physiological studies in rainbow trout. Interestingly, we found that the rainbow trout MC4 receptor has a natural high-affinity binding site for zinc ions (0.5 microM) indicating that zinc may play an evolutionary conserved role at this receptor. Reverse transcription PCR indicates that the rainbow trout receptors are expressed both in peripheral tissues and in the central nervous system, including the telencephalon, optic tectum and hypothalamus. Overall, this analysis indicates that the rainbow trout MC4 and MC5 receptors have more in common than their mammalian counterparts, which may suggest that these two receptors have a closer evolutionary relationship than the other MC receptor subtypes.

Human genes for an adrenocorticotropin (ACTH) receptor and a melanocyte-stimulating hormone (MSH) receptor, and a mouse gene for a MSH receptor have been cloned recently. They constitute a family of receptors for peptides derived from proopiomelanocortin. The ACTH receptor mRNA and the MSH receptor mRNA were expressed only in the adrenal and melanocytes, respectively. Thereafter, three novel receptor genes for melanocortins were molecularly cloned and were expressed exclusively in the brain but not in the adrenal or melanocytes. It has remained unknown how these receptors are differentially expressed in specialized cell type. An adrenocortical cell line of mouse origin, Y-1, is to date the sole cell line which shows responsiveness to ACTH resulting in the induction of steroids. For analysis of the transcriptional regulation of ACTH receptor in Y-1 cells, I have isolated the mouse ACTH receptor gene. A 914 bp DNA fragment of the human ACTH receptor gene was amplified by polymerase chain reaction and used as a probe. Eight positive clones were obtained by screening 10(6) phage clones of a mouse genomic library under the condition of low stringency. One phage clone, lambda mCTR8 which gave the most intense hybridization signal was analyzed. It contained an approximately 12 kb insert. By comparing it with the previously reported human ACTH receptor gene and bovine ACTH receptor cDNA, the mouse homologue is intron-less in its coding region, and has a potential to encode for a 296-amino-acid polypeptide which possesses 88.9% and 78.7% identities to the human and bovine ACTH receptors, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A mouse genomic clone named HGMP01B has been isolated by homology screening with a probe representing part of the human melanocortin 3 receptor gene. HGMP01B was found to encode a 325 amino acid protein with all the landmarks of G-protein-coupled receptors and belonging to the growing melanocortin receptor family. This receptor displays four potential sites for N-linked glycosylation and five potential sites of phosphorylation by protein kinase C. The HGMP01B gene was found to be expressed in many tissues, including skin, adrenal gland, skeletal muscle, bone marrow, spleen, thymus, gonads, uterus, and brain. A stable Chinese hamster ovary (CHO) cell line expressing approximately 10,000 receptors per cell was established. This cell line displayed a saturable binding capacity for the radioiodinated alpha-melanocyte-stimulating hormone (alpha-MSH) analog [Nle4,D-Phe7]-alpha-MSH (NDP-MSH) with an apparent Kd of 1.47 +/- 0.15 nM. Binding of the labeled ligand was competed for by all melanocortin peptides, except beta-endorphin or corticotropin-like intermediate lobe peptide (CLIP). NDP-MSH was the most powerful competitor, followed by alpha-MSH, adrenocorticotropic hormone (ACTH), beta-MSH, the gamma-MSHs, and ACTH 4-10. Functional assays confirmed that HGMP01B, like other melanocortin receptors, stimulated adenylyl cyclase. The potency order obtained in these cyclic adenosine monophosphate (cAMP) accumulation assays was consistent with that of the binding studies. HGMP01B therefore appears as a fifth melanocortin receptor (MC5), responding mainly to alpha-MSH (EC50 = 1.07 +/- 0.13 nM) and endowed with a pharmacological profile similar to that of the melanocyte MSH (MC1) receptor, but characterized by a broad tissue distribution.(ABSTRACT TRUNCATED AT 250 WORDS)