Gαi and Gβγ Jointly Regulate the Conformations of a Gβγ Effector, the Neuronal G Protein-activated K+ Channel (GIRK)

Shai Berlin,Tal Keren-Raifman,Ruth Castel,Moran Rubinstein,Carmen W Dessauer,Tatiana Ivanina,Nathan Dascal

doi:10.1074/jbc.m109.085944

Abstract

Stable complexes among G proteins and effectors are an emerging concept in cell signaling. The prototypical G betagamma effector G protein-activated K(+) channel (GIRK; Kir3) physically interacts with G betagamma but also with G alpha(i/o). Whether and how G alpha(i/o) subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, G alpha(i3) and G betagamma. We used in vitro protein interaction assays and in vivo intramolecular Förster resonance energy transfer (i-FRET) between fluorophores attached to N and C termini of either GIRK1 or GIRK2 subunit. We demonstrate, for the first time, that G betagamma and G alpha(i3) distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that G betagamma greatly enhances the binding of GIRK1 subunit to G alpha(i3)(GDP) and, unexpectedly, to G alpha(i3)(GTP). i-FRET showed that both G alpha(i3) and G betagamma induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct conformations when coexpressed with a "constitutively active" G alpha(i3) mutant and G betagamma together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either G alpha(i3) or G betagamma. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-G alpha-G betagamma signaling complex and mediates allosteric interactions between G alpha(i)(GTP) and G betagamma. Our findings imply that G alpha(i/o) and the G alpha(i) betagamma heterotrimer can regulate a G betagamma effector both before and after activation by neurotransmitters.

Highlights

It is believed that signaling via G protein-coupled receptors (GPCRs)5 occurs within multiprotein complexes that include
We proposed that regulation of GIRK by G␣i relies upon the formation of the G␣iGDP-G␤␥ heterotrimer, which forms a persistent, dynamic signaling complex with GIRK to ensure proper gating with low i reduces GIRK basal activity (Ibasal) and high signal-to-background ratio upon G␤␥ activation [11, 12]
Somewhat at odds with Huang et al [5], our data suggest that both NT and CT are necessary for the formation of the strong GIRK1-G␣i␤␥ complex as the effect of G␤␥ was not present in separate N and C termini

Summary

EXPERIMENTAL PROCEDURES

Additional details on all methods are available in the supplemental methods. cDNA Constructs and Electrophysiology—The cDNAs used in this study were obtained or prepared using standard PCRbased procedures. A GST-fused G␣i3 bound ivt G1NC as well as separate G1C and G1NYFP, whereas YFP or the distal CT (G1C363–501) did not show detectable binding (Fig. 1, B and C). The latter observations confirm results obtained in a reciprocal configuration, with GST-fused channel parts and ivt G␣i3 [9]. The addition of purified G␤1␥2 had no effect on the binding of GST-G␣i3 to separate GIRK1 N and C termini, in the presence of either GDP or GTP␥S (Fig. 1B, summary in panel C). GIRK1 likely binds the active G␣i3 (G␣i3GTP␥S and G␣i3QL) directly and not through G␤␥; by binding to GIRK1, G␤␥ enhances this interaction

Findings

G Protein Subunits Induce Non-identical Conformational

DISCUSSION