Preactivation permits subsequent stimulation of phospholipase C by G(i)-coupled receptors.
In the complex signal transduction networks involving G protein-coupled receptors there are numerous examples where G(i)-linked receptors augment G(q)-dependent signals. The mechanistic basis of such occurrences is thought to entail signal convergence at phospholipase Cbeta (PLCbeta) via the G protein betagamma-dimers. Herein, we explored the possibility that augmentation by betagamma-dimers requires preactivation of PLCbeta. COS-7 cells were transiently cotransfected with cDNAs encoding various combinations of receptors and G protein subunits. The G(i)-coupled delta- and kappa-opioid receptors could not stimulate PLCbeta unless they were coexpressed with Galpha(16). The opioid-induced response was dose-dependent and partially inhibited by pertussis toxin or coexpression with transducin, indicating the involvement of betagamma-subunits released from the G(i) proteins. When PLCbeta was preactivated by constitutively active mutants of Galpha(16), Galpha(q), or Galpha(14), opioids enhanced the activity by 80 to 300% and such responses were mostly pertussis toxin-sensitive. The opioid-induced enhancement was dose-dependent and could not be blocked by staurosporin, a protein kinase C inhibitor. Other G(i)-coupled receptors that were ineffective on their own also acquired the ability to stimulate PLCbeta in the presence of a constitutively active mutant of Galpha(q). Coactivation of endogenous or exogenous G(q)-coupled receptors with the delta-opioid receptor produced strong stimulations of PLCbeta and such responses could be partially blocked by pertussis toxin. These results show that enhancement of G(q)-dependent signals by G(i)-coupled receptors requires activated PLCbeta and is mediated via the betagamma-dimer.
Chan
J S
JS
Department of Biology and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
Lee
J W
JW
Ho
M K
MK
Wong
Y H
YH
D000818:Animals; D019556:COS Cells; D004789:Enzyme Activation; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D037342:Pertussis Toxin; D054799:Phospholipase C beta; D010738:Type C Phospholipases / Q000187:drug effects / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007527:Isoenzymes; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D054799:Phospholipase C beta; C112631:G protein alpha 16; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins