Abstract
RGS3 and RGS4 are GTPase-activating proteins expressed in the brain and heart that accelerate the termination of G(i/o)- and G(q)-mediated signaling. We report here the determinants mediating selective association of RGS4 with several G protein-coupled receptors (GPCRs) that form macromolecular complexes with neuronal G protein-gated inwardly rectifying potassium (Kir3 or GIRK) channels. Kir3 channels are instrumental in regulating neuronal firing in the central and peripheral nervous system and pacemaker activity in the heart. By using an epitope-tagged degradation-resistant RGS4 mutant, RGS4(C2V), immunoprecipitation of several hemagglutinin-tagged G(i/o)-coupled and G(q)-coupled receptors expressed in Chinese hamster ovary (CHO-K1) cells readily co-precipitated both Kir3.1/Kir3.2a channels and RGS4(C2V). In contrast to RGS4(C2V), the closely related and functionally active RGS3 "short" isoform (RGS3s) did not interact with any of the GPCR-Kir3 channel complexes examined. Deletion and chimeric RGS constructs indicate both the N-terminal domain and the RGS domain of RGS4(C2V) are necessary for association with m2 receptor-Kir3.1/Kir3.2a channel complexes, where the GPCR was found to be the major target for RGS4(C2V) interaction. The functional impact of RGS4(C2V) "precoupling" to the GPCR-Kir3 channel complex versus RGS3s "collision coupling" was a 100-fold greater potency in the acceleration of G protein-dependent Kir3 channel-gating kinetics with no attenuation in current amplitude. These findings demonstrate that RGS4, a highly regulated modulator and susceptibility gene for schizophrenia, can directly associate with multiple GPCR-Kir3 channel complexes and may affect a wide range of neurotransmitter-mediated inhibitory and excitatory events in the nervous and cardiovascular systems.
Highlights
Our findings demonstrate a strong association between RGS4(C2V) and several GPCRs that are central participants in normal and pathologically altered neuromodulation of membrane excitability
Given the multiple mechanisms affecting RGS4 protein levels, including the recently described impact of the oxidative environment (26), it will be important to determine to what extent these changes in RGS4 concentration and modification affect coupling to different GPCR signaling pathways
Acquired or inherited disruptions in RGS4-GPCR coupling may play a role in a variety of neurological disorders that include schizophrenia (12), Parkinson disease (73), depression, epilepsy, and drug addiction (64)
Summary
N-terminal Tagged GPCRs—Complementary DNAs encoding the human muscarinic m2 receptor (GenBankTM accession number NM_000739), human serotonin 1A receptor (GenBankTM accession number NM_000524), and mouse lysophosphatidic acid (LPA1/edg2) receptor (GenBankTM accession number NM_010336) were “tagged” at their N termini with the HA sequence (YPYDVPDYA). The complete coding region of the human muscarinic m2 receptor, human serotonin 1A receptor, and mouse LPA1 receptor were amplified by PCR and cloned in-frame at the XbaI site of the N-terminal HA tag pcDNA3.1(ϩ) vector. The human adenosine A1 receptor (GenBankTM accession number AY136746), human dopamine D2L receptor (GenBankTM accession number NM_000795), and human muscarinic m1 receptor (GenBankTM accession number AF498915) were obtained from the University of Missouri, Rolla cDNA Resource Center, and contained N-terminal triple (3ϫ) HA tags, and were cloned into the pcDNA3.1(ϩ) vector
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