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.

Periplakin interferes with G protein activation by the melanin-concentrating hormone receptor-1 by binding to the proximal segment of the receptor C-terminal tail.

In mice genetic ablation of expression of either melanin-concentrating hormone or the melanin-concentrating hormone-1 receptor results in alterations in energy metabolism and a lean phenotype. There is thus great interest in the function and regulation of this receptor. Using the yeast two-hybrid system we identified an interaction of the actin- and intermediate filament-binding protein periplakin with the intracellular C-terminal tail of the melanin-concentrating hormone-1 receptor. Direct association of these proteins was verified in pull-down and coimmunoprecipitation experiments. Truncations and internal deletions delineated the site of interaction to a group of 11 amino acids proximal to transmembrane helix VII, which was distinct from the binding site for the melanin-concentrating hormone-1 receptor-interacting zinc finger protein. Immunohistochemistry demonstrated coexpression of periplakin with melanin-concentrating hormone-1 receptor in specific cells of the piriform cortex, amygdala, and other structures of the adult mouse brain. Coexpression of the melanin-concentrating hormone-1 receptor with periplakin in human embryonic kidney 293 cells did not prevent agonist-mediated internalization of the receptor but did interfere with binding of (35)S-labeled guanosine 5'-3-O-(thio)triphosphate ([(35)S]GTPgammaS) to the G protein Galpha(o1) and the elevation of [Ca(2+)](i). Coexpression of the receptor with the interacting zinc finger protein did not modulate receptor internalization or G protein activation. The interaction of periplakin with receptors was selective. Coexpression of periplakin with the IP prostanoid receptor did not result in coimmunoprecipitation nor interfere with agonist-mediated binding of [(35)S]GTPgammaS to the G protein Galpha(s). Periplakin is the first protein described to modify the capacity of the melanin-concentrating hormone-1 receptor to initiate signal transduction.

Functional annotation of two orphan G-protein-coupled receptors, Drostar1 and -2, from Drosophila melanogaster and their ligands by reverse pharmacology.

By combining a Drosophila genome data base search and reverse transcriptase-PCR-based cDNA isolation, two G-protein-coupled receptors were cloned, which are the closest known invertebrate homologs of the mammalian opioid/somatostatin receptors. However, when functionally expressed in Xenopus oocytes by injection of Drosophila orphan receptor RNAs together with a coexpressed potassium channel, neither receptor was activated by known mammalian agonists. By applying a reverse pharmacological approach, the physiological ligands were isolated from peptide extracts from adult flies and larvae. Edman sequencing and mass spectrometry of the purified ligands revealed two decapentapeptides, which differ only by an N-terminal pyroglutamate/glutamine. The peptides align to a hormone precursor sequence of the Drosophila genome data base and are almost identical to allatostatin C from Manduca sexta. Both receptors were activated by the synthetic peptides irrespective of the N-terminal modification. Site-directed mutagenesis of a residue in transmembrane region 3 and the loop between transmembrane regions 6 and 7 affect ligand binding, as previously described for somatostatin receptors. The two receptor genes each containing three exons and transcribed in opposite directions are separated by 80 kb with no other genes predicted between. Localization of receptor transcripts identifies a role of the new transmitter system in visual information processing as well as endocrine regulation.