Elsevier

Cellular Signalling

Volume 18, Issue 3, March 2006, Pages 336-348
Cellular Signalling

RGS2 interacts with Gs and adenylyl cyclase in living cells

https://doi.org/10.1016/j.cellsig.2005.05.004Get rights and content

Abstract

Regulator of G Protein Signalling (RGS) proteins impede heterotrimeric G protein signalling. RGS2 decreases cAMP production and appears to interact with both adenylyl cyclase (AC) and its stimulatory G protein Gs. We showed previously that Green Fluorescent Protein-tagged RGS2 (GFP-RGS2) localizes to the nucleus in HEK 293 cells and is recruited to the plasma membrane when co-expressed with Gsα, or the Gs-coupled β2-adrenergic receptor (β2AR). Here, using confocal microscopy we show that co-expression of various AC isoforms (ACI, ACII, ACV, ACVI) also leads to GFP-RGS2 recruitment to the plasma membrane. Bioluminescence Resonance Energy Transfer (BRET) was also used to examine physical interactions between RGS2 and components of the Gs-signalling pathway. A BRET signal was detected between fusion constructs of RGS2-Renilla luciferase (energy donor) and Gsα-GFP (energy acceptor) co-expressed in HEK 293 cells. BRET was also observed between GFP-RGS2 and ACII or ACVI fused to Renilla luciferase. Additionally, RGS2 was found to interact with the β2AR. Purified RGS2 selectively bound to the third intracellular loop of the β2AR in GST pulldown assays, and a BRET signal was observed between GFP-RGS2 and β2AR fused to Renilla luciferase when these two proteins were co-expressed together with either ACIV or ACVI. This interaction was below the limit of detection in the absence of co-expressed AC, suggesting that the effector enzyme stabilized or promoted binding between the receptor and the RGS protein inside the cell. Taken together, these results suggest the possibility that RGS2 might bind to a receptor-G protein-effector signalling complex to regulate Gs-dependent cAMP production.

Introduction

G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by promoting the dissociation of bound GDP and the association of the activating nucleotide GTP. Both the Gα subunit and the Gβγ dimer of the activated G protein heterotrimer are able to interact with effectors in the plasma membrane, and signalling is terminated by intrinsic hydrolysis of GTP by the Gα subunit. RGS proteins are GTPase-activating proteins (GAPs) that increase the rate of hydrolysis of GTP by Gα subunits. Approximately 20 different RGS proteins, not including splice variants and “RGS-like” proteins, have been identified. However, selectivity for different G protein subfamilies is limited in that most RGS proteins interact with the Gi family of G proteins, with a subset also capable of interacting with Gq [1], [2].

Although RGS proteins do not seem to act as GAPs for Gs [3], [4], there is evidence for RGS protein/Gs interactions. We have recently demonstrated that RGS2 is recruited from the nucleus to the plasma membrane of HEK 293 cells upon co-expression of Gsα [4]. RGS2 has also been shown to attenuate Gs-stimulated increases in intracellular cAMP levels [4], [5], [6], [7], [8]. The C1 domain of AC type V binds purified RGS2, providing a possible mechanism for the inhibitory effects of RGS2 on intracellular cAMP accumulation [6]. However, other evidence suggests that these inhibitory effects may reflect physical interactions between RGS2 and Gs, since RGS2 binds to Gs-GDP in a fluoroaluminate-sensitive manner [9], and similarly has been co-immunoprecipitated with Gs from cells upon the activation of a Gs-coupled receptor [8]. Signalling via the β2-adrenergic receptor (β2AR) is also dependent upon the activation of Gs and adenylyl cyclase, and we have recently demonstrated that co-expression of the β2AR also facilitates GFP-RGS2 recruitment to the plasma membrane of HEK 293 cells [4].

GPCR signalling has historically been described as the result of random collisions between interacting proteins occurring at or near the plasma membrane. The use of in vivo interaction assays based on bioluminescence or fluorescence resonance energy transfer (BRET/FRET) has altered this perception. BRET has been used to screen for physical interactions between GPCRs, G protein subunits and their effectors. A number of studies have demonstrated that interactions between the above mentioned proteins are stable, and persist during signal transduction (reviewed in [10]). For example, the β2AR has been demonstrated to form stable homo-[11] and heterodimers (with both β1AR and β3AR) [12], [13], and to stably interact with its effector enzyme adenylyl cyclase [14]. Taken together, these data suggest the possible existence of stable multicomponent Gs-coupled signalling complexes localized to the plasma membrane.

We have used BRET to determine whether direct interactions between RGS2 and Gs, adenylyl cyclase or the β2AR occur in living cells. We show that RGS2 interacts with both Gsα and several adenylyl cyclase isoforms at the plasma membrane of HEK 293 cells, to regulate and limit Gs-mediated signalling.

Section snippets

DNA constructs

The construction of pEGFP-C3-mRGS2 has been described by us previously [4]. pcDNA3-β2AR-EGFP was a gift from J. Benovic (Thomas Jefferson University). pGL3-Basic encoding firefly luciferase was purchased from Promega. pEBG2 encoding Glutathione-S-Transferase (GST), Flag-β2AR and pRLuc-N2 were generously donated by S. Ferguson (Robarts Research Institute). pcDNA3-mRGS2 was generously donated by D. Siderovski (University of North Carolina). Human Gsα-Long expression vector was purchased from the

Co-expression of adenylyl cyclase alters the intracellular localization of RGS2

GFP-RGS2 was expressed in HEK 293 cells and as expected demonstrated a predominantly nuclear localization [4]. In contrast, when GFP-RGS2 was co-expressed with either adenylyl cyclase type I, II, V or VI, GFP-RGS2 was recruited to the plasma membrane compartment in the majority of cells examined (Fig. 1). One possible explanation for the recruitment of GFP-RGS2 to the plasma membrane by adenylyl cyclase is that the two proteins interact. Further experiments thus were directed towards examining

Discussion

We sought to determine which potential binding partners might be responsible for plasma membrane recruitment of GFP-RGS2 by components of the β-adrenergic signalling system. GFP-RGS2 was recruited from the nucleus to the plasma membrane upon co-expression of all adenylyl cyclase subtypes tested. To investigate the possibility that RGS2 might interact with adenylyl cyclase, HEK 293 cells were co-transfected with GFP-RGS2 and various adenylyl cyclase isoforms. Our results indicate that RGS2 binds

Conclusions

Previous work has suggested that RGS2 may produce its inhibitory effects on Gs-mediated adenylyl cyclase activity via binding to Gsα or adenylyl cyclase. The present results would appear to resolve this discrepancy, as we found that RGS2 can bind to both of these proteins. Since the G protein and the effector protein did not appear to compete for binding to RGS2, it follows that RGS2 may attenuate cAMP production by binding to both of these proteins within a heteromeric signalling complex.

Acknowledgements

This work was supported by grants from the Canadian Institutes of Health Research to P.C. and T.E.H. and Heart and Stroke Foundation of Quebec to T.E.H. T.E.H. is a MacDonald Scholar of the Heart and Stroke Foundation of Canada. A.A.R. is supported by the Natural Sciences and Engineering Research Council of Canada and the Heart and Stroke Foundation of Canada. A.B. holds a Government of Canada award in conjunction with the Government of Italy. We thank R.V. Rebois for helpful discussions, and

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