Preactivation Permits Subsequent Stimulation of Phospholipase C by Gi-Coupled Receptors
- Department of Biology and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
Abstract
In the complex signal transduction networks involving G protein-coupled receptors there are numerous examples where Gi-linked receptors augment Gq-dependent signals. The mechanistic basis of such occurrences is thought to entail signal convergence at phospholipase Cβ (PLCβ) via the G protein βγ-dimers. Herein, we explored the possibility that augmentation by βγ-dimers requires preactivation of PLCβ. COS-7 cells were transiently cotransfected with cDNAs encoding various combinations of receptors and G protein subunits. The Gi-coupled δ- and κ-opioid receptors could not stimulate PLCβ unless they were coexpressed with Gα16. The opioid-induced response was dose-dependent and partially inhibited by pertussis toxin or coexpression with transducin, indicating the involvement of βγ-subunits released from the Giproteins. When PLCβ was preactivated by constitutively active mutants of Gα16, Gαq, or Gα14, 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 Gi-coupled receptors that were ineffective on their own also acquired the ability to stimulate PLCβ in the presence of a constitutively active mutant of Gαq. Coactivation of endogenous or exogenous Gq-coupled receptors with the δ-opioid receptor produced strong stimulations of PLCβ and such responses could be partially blocked by pertussis toxin. These results show that enhancement of Gq-dependent signals by Gi-coupled receptors requires activated PLCβ and is mediated via the βγ-dimer.
Footnotes
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Send reprint requests to: Yung H. Wong, Department of Biology and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. E-mail: boyung{at}ust.hk
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This work was supported by the Research Grants Council of Hong Kong (HKUST 653/96M and HKUST 6096/98M) and the Hong Kong Jockey Club.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- PLCβ
- phospholipase Cβ
- IP
- inositol phosphate
- PTX
- pertussis toxin
- fMLP
- formyl peptide
- PCR
- polymerase chain reaction
- DMEM
- Dulbecco's modified Eagle's medium
- DOR
- δ-opioid receptor
- DPDPE
- [d-Pen2,5]enkephalin
- ORL
- opioid receptor-like
- KOR
- κ-opioid receptor
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- Received July 23, 1999.
- Accepted December 20, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
