Differential modulation of mu-opioid receptor signaling to adenylyl cyclase by regulators of G protein signaling proteins 4 or 8 and 7 in permeabilised C6 cells is Galpha subtype dependent.
Regulators of G protein signaling (RGS) proteins act as GTPase-accelerating protein to negatively modulate G protein signaling and are defined by a conserved RGS domain with considerable amino acid diversity. To determine the effects of specific, purified RGS proteins on mu-opioid signaling, C6 cells stably expressing a mu-opioid receptor were rendered permeable to proteins by treatment with digitonin. Mu-opioid inhibition of forskolin-stimulated adenylyl cyclase by [D-Ala(2),N-Me-Phe(4),Gly-ol]-enkephalin (DAMGO), a mu-specific opioid peptide, remained fully intact in permeabilized cells. Purified RGS domain of RGS4 added to permeabilized cells resulted in a twofold loss in DAMGO potency but had no effect in cells expressing RGS-insensitive G proteins. The inhibitory effect of DAMGO was reduced to the same extent by purified RGS4 and RGS8. In contrast, the RGS domain of RGS7 had no effect and inhibited the action of RGS8 as a result of weak physical association with Galphai2 and minimal GTPase-accelerating protein activity in C6 cell membranes. These data suggest that differences in conserved RGS domains of specific RGS proteins contribute to differential regulation of opioid signaling to adenylyl cyclase and that a permeabilized cell model is useful for studying the effects of specific RGS proteins on aspects of G protein-coupled receptor signaling.
Talbot
Jeffery N
JN
Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA.
Roman
David L
DL
Clark
Mary J
MJ
Roof
Rebecca A
RA
Tesmer
John J G
JJ
Neubig
Richard R
RR
Traynor
John R
JR
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D045744:Cell Line, Tumor; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology*; D005910:Glioma; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D020710:RGS Proteins / Q000378:metabolism*; D051381:Rats; D017450:Receptors, Opioid, mu / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D000701:Analgesics, Opioid; D020710:RGS Proteins; D017450:Receptors, Opioid, mu; C115771:Rgs7 protein, rat; C486485:Rgs8 protein, rat; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; C098799:RGS4 protein; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D004072:Digitonin