Selective interactions between helix VIII of the human mu-opioid receptors and the C terminus of periplakin disrupt G protein activation.
Analysis of interactions between the C-terminal tail of the MOP-1 and MOP-1A variants of the human mu-opioid receptor with proteins derived from a human brain cDNA library resulted in identification of the actin and intermediate filament-binding protein periplakin. Mapping of this interaction indicated that the predicted fourth intracellular loop/helix VIII of the receptor interacts with the C-terminal rod and linker region of periplakin. Periplakin is widely expressed in the central nervous system of both man and rat and demonstrated an overlapping but not identical distribution with mu-opioid (MOP) receptors. Co-expression of periplakin with MOP-1 or a MOP-1-eYFP fusion construct in HEK293 cells did not interfere with agonist-mediated internalization of the receptor. When co-expressed with a MOP-1-Gi1 alpha fusion protein periplakin significantly reduced the capacity of the agonist to stimulate binding of 35S-labeled guanosine 5'-3-O-(thio)triphosphate ([35S]GTP gamma S) to the receptor-associated G protein. By contrast, periplakin did not interfere with agonist-stimulation of [35S]GTP gamma S binding to either an alpha 2A-adrenoreceptor-Gi1 alpha fusion protein or a beta2-adrenoreceptor-Gs alpha fusion protein, indicating its selectivity of function. This represents the first example of an opioid receptor-interacting protein that functions to disrupt agonist-mediated G protein activation.
Feng
Giu-Jie
GJ
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
Kellett
Elaine
E
Scorer
Carol A
CA
Wilde
Jonathan
J
White
Julia H
JH
Milligan
Graeme
G
D000199:Actins / Q000737:chemistry; D000595:Amino Acid Sequence; D001710:Biotin / Q000494:pharmacology; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002490:Central Nervous System / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry*; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D051179:Plakins; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014162:Transfection; D020798:Two-Hybrid System Techniques
D000199:Actins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D008024:Ligands; C491674:PPL protein, human; D051179:Plakins; D020033:Protein Isoforms; D012333:RNA, Messenger; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006639:Histidine; D001710:Biotin; D004247:DNA; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins