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.
Francke
Felix
F
Institute for Cell Biochemistry and Clinical Neurobiology, University Hospital Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
Ward
Richard J
RJ
Jenkins
Laura
L
Kellett
Elaine
E
Richter
Dietmar
D
Milligan
Graeme
G
Bächner
Dietmar
D
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011979:Receptors, Pituitary Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D011979:Receptors, Pituitary Hormone; D011993:Recombinant Fusion Proteins; C092274:melanin-concentrating hormone receptor; C119189:neurochondrin; D019204:GTP-Binding Proteins