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.
Murdoch
Hannah
H
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
Feng
Gui-Jie
GJ
Bächner
Dietmar
D
Ormiston
Laura
L
White
Julia H
JH
Richter
Dietmar
D
Milligan
Graeme
G
D000199:Actins / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry*; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D010641:Phenotype; D010766:Phosphorylation; D051179:Plakins; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017481:Receptors, Somatostatin / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers
D000199:Actins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; C103263:MCHR1 protein, human; D051179:Plakins; C110037:Ppl protein, mouse; D012333:RNA, Messenger; D017481:Receptors, Somatostatin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006639:Histidine; D012313:RNA; D001710:Biotin; D004247:DNA; D005982:Glutathione Transferase; D048049:Extracellular Signal-Regulated MAP Kinases; D048052:Mitogen-Activated Protein Kinase 3; D019204:GTP-Binding Proteins; D002118:Calcium