Selective interactions between helix VIII of the human μ-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-G(i1)alpha fusion protein periplakin significantly reduced the capacity of the agonist to stimulate binding of S-35-labeled guanosine 5'-3-O-(thio)triphosphate ([S-35]GTPgammaS) to the receptor-associated G protein. By contrast, periplakin did not interfere with agonist-stimulation of [S-35]GTPgammaS binding to either an alpha(2A)-adrenoreceptor-G(i1)alpha fusion protein or a beta(2)-adrenoreceptor-G(s)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.