Identification of mRNAs coding for mammalian-type melanin-concentrating hormone and its receptors in the scalloped hammerhead shark Sphyrna lewini.

Melanin-concentrating hormone (MCH) is a neuromodulator, synthesized in the hypothalamus, that regulates both appetite and energy homeostasis in mammals. MCH was initially identified in teleost fishes as a pituitary gland hormone that induced melanin aggregation in chromatophores in the skin; however, this function of MCH has not been observed in other vertebrates. Recent studies suggest that MCH is involved in teleost feeding behavior, spurring the hypothesis that the original function of MCH in early vertebrates was appetite regulation. The present study reports the results of cDNAs cloning encoding preproMCH and two MCH receptors from an elasmobranch fish, Sphyrna lewini, a member of Chondrichthyes, the earliest diverged class in gnathostomes. The putative MCH peptide is composed of 19 amino acids, similar in length to the mammalian MCH. Reverse-transcription polymerase chain reaction revealed that MCH is expressed in the hypothalamus in S. lewini MCH cell bodies and fibers were identified by immunochemistry in the hypothalamus, but not in the pituitary gland, suggesting that MCH is not released via the pituitary gland into general circulation. MCH receptor genes mch-r1 and mch-r2 were expressed in the S. lewini hypothalamus, but were not found in the skin. These results indicate that MCH does not have a peripheral function, such as a melanin-concentrating effect, in the skin of S. lewini hypothalamic MCH mRNA levels were not affected by fasting, suggesting that feeding conditions might not affect the expression of MCH in the hypothalamus.

Interaction of neurochondrin with the melanin-concentrating hormone receptor 1 interferes with G protein-coupled signal transduction but not agonist-mediated internalization.

Screening of a human brain cDNA library using the C-terminal tail of the melanin-concentrating hormone receptor 1 (MCHR1) as bait in a yeast two-hybrid assay resulted in the identification of the neurite-outgrowth related factor, neurochondrin. This interaction was verified in overlay, pulldown, and co-immunoprecipitation assays. Deletion mapping confined the binding to the C terminus of neurochondrin and to the proximal C terminus of MCHR1, a region known to be involved in G protein binding and signal transduction. This region of the MCHR1 is also able to interact with the actin- and intermediate filament-binding protein, periplakin. Interactions of MCHR1 with neurochondrin and periplakin were competitive, indicating that these two proteins bind to overlapping regions of MCHR1. Although neurochondrin did not interfere with melanin-concentrating hormone-mediated internalization of the receptor, it did inhibit G protein-coupled signal transduction via both Galpha(i/o) and Galpha(q/11) family G proteins as measured by each of melanin-concentrating hormone-induced G protein-activated inwardly rectifying K(+) channel activity of voltage-clamped amphibian oocytes, by calcium mobilization in transfected mammalian cells, and by reduction in the capacity of melanin-concentrating hormone to promote binding of [(35)S]guanosine 5'-3-O-(thio)triphosphate to both Galpha(o1) and Galpha(11). Immunohistochemistry revealed co-expression of neurochondrin and MCHR1 within the rodent brain, suggesting that neurochondrin may be involved in the regulation of MCHR1 signaling and play a role in modulating melanin-concentrating hormone-mediated functions in vivo.

Role of second extracellular loop in the function of human vasoactive intestinal polypeptide/pituitary adenylate cyclase activating polypeptide receptor 1 (hVPAC1R).

To elucidate the functional role of the second extracellular loop of human vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide (VIP/PACAP) receptor (hVPAC1R), surface expression, ligand binding, and receptor activation were analyzed. Amino acids in the entire second extracellular loop were individually substituted by alanine by site-directed mutagenesis. The mutant and wild-type receptors were transiently expressed in HEK293 cells and purified cell membranes were tested for the ability to bind VIP, while the receptor activity was measured as potency of cAMP production analysed on intact cells. Surface expression of the substituted conserved residues, W286A, I289A, W294A, and W295A, was evidently decreased to 20-30% compared to the wild-type expression. W286A also showed an significantly reduced potency of cAMP production. Substituted residues as F280A, E281A, and G284A showed a significant reduction in the potency of stimulated cAMP production amounting to 8-46-fold, compared to the wild-type with unaffected surface expression and VIP binding. These results indicate that some residues in the second extracellular loop of the human VPAC1R participate in the active mechanism of a ligand-mediated response without being directly involved in the binding of VIP.

Differential signaling and immediate-early gene activation by four splice variants of the human pituitary adenylate cyclase-activating polypeptide receptor (hPACAP-R).

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide belonging to the VIP/secretin/glucagon family, is present in the hypothalamus, anterior pituitary, and adrenal gland where it regulates hormone release, in the GI tract where it modulates motility, and in human tumoral cell lines where it shows a growth-promoting effect. It is now appreciated that alternative splicing of two exons of the rat PACAP-R gene generate four major rPACAP-R splice variants that are differentially expressed in tissues and variably coupled to intracellular second messengers. Because of the potential implications of these findings in human physiology, we cloned the hPACAP-R gene. Similar to the rat, two exons (SV-1 and SV-2) are alternatively spliced to account for four major hPACAP-R receptor splice variants. These splice variants (hPACAP-R-null, hPACAP-R-SV1, hPACAP-R-SV2, hPACAP-R-SV-3) were cloned from a human frontal cortex cDNA library, stably transfected in NIH/ 3T3 cells and each characterized for ligand affinity, stimulation of adenylate cyclase (AC) and phospholipase C (PLC), and ligand-induced expression of the proto-oncogenes, c-fos, and c-myc. Stably transfected NIH/3T3 cells expressing similar numbers of receptors of the four splice variants showed nearly identical responses for ligand affinity and potency for P-38- and P-27-stimulated increases in cAMP and total inositol phosphates. However, each receptor splice variant differed in their ligand-stimulated efficacy for total inositol phosphate stimulation. The hPACAP-R-SV2 showed the greatest efficacy for stimulating phospholipase C that was approximately seven-fold greater than the hPACAP-R-SV1, twofold greater than the hPACAP-R-Null, and 1.5-fold greater than the hPACAP-R-SV-3 splice variants. To determine whether the splice variants also differ in their ability to stimulate immediate early gene expression, c-fos and c-myc transcripts were assayed by Northern blot and quantified by densitometry. PACAP-38 increased c-fos and c-myc expression for all four of the receptor splice variants that paralleled the efficacy for PLC stimulation, with the the SV-2 splice variant showing the greatest stimulation. These results show that the hPACAP-R-SV2 exhibits enhanced efficacy for coupling to both PLC and activation of the protooncogenes, c-fos and c-myc suggesting a novel and potentially important mechanism for differentially activating signal transduction pathways that influence cellular growth and differentiation.

Identification of a receptor for gamma melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system.

Corticotropin (ACTH) and melanotropin (MSH) peptides (melanocortins) are produced not only in the pituitary but also in the brain, with highest concentrations in the arcuate nucleus of the hypothalamus and the commisural nucleus of the solitary tract. We have identified a receptor for MSH and ACTH peptides that is specifically expressed in regions of the hypothalamus and limbic system. This melanocortin receptor (MC3-R) is found in neurons of the arcuate nucleus known to express proopiomelanocortin (POMC) and in a subset of the nuclei to which these neurons send projections. The MC3-R is 43% identical to the MSH receptor present in melanocytes and is strongly coupled to adenylyl cyclase. Unlike the MSH or ACTH receptors, MC3-R is potently activated by gamma-MSH peptides, POMC products that were named for their amino acid homology with alpha- and beta-MSH, but lack melanotropic activity. The primary biological role of the gamma-MSH peptides is not yet understood. The location and properties of this receptor provide a pharmacological basis for the action of POMC peptides produced in the brain and possibly a specific physiological role for gamma-MSH.