Characterization of the Gαs Regulator Cysteine String Protein

Michael Natochin,Shahid Hameed,Janice E.A Braun,Brandy Barren,Linda C Miller,Nikolai O Artemyev,Tessa N Campbell

doi:10.1074/jbc.m500722200

Michael Natochin, Shahid Hameed + Show 5 more

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https://doi.org/10.1074/jbc.m500722200

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Abstract

Cysteine string protein (CSP) is an abundant regulated secretory vesicle protein that is composed of a string of cysteine residues, a linker domain, and an N-terminal J domain characteristic of the DnaJ/Hsp40 co-chaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca2+ channels. To elucidate the mechanisms by which CSP modulates G protein signaling, we examined the effects of CSP1–198 (full-length), CSP1–112, and CSP1–82 on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with Gαs and increases steady-state GTP hydrolysis. CSP1–198 modulation of Gαs was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP1–112 was Hsc70-SGT-independent. CSP1–112 preferentially associated with the inactive GDP-bound conformation of Gαs. Consistent with the stimulation of GTP hydrolysis, CSP1–112 increased guanine nucleotide exchange of Gαs. The interaction of native Gαs and CSP was confirmed by coimmunoprecipitation and showed that Gαs associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the β2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of Gαs and promoting GDP/GTP exchange. Our results show that the guanine nucleotide exchange activity of full-length CSP is, in turn, regulated by Hsc70-SGT.

Highlights

G proteins constitute a family of heterotrimeric GTP-binding proteins that act as transducers in a variety of transmembrane signaling systems
To elucidate the mechanisms by which Cysteine string protein (CSP) modulates G protein signaling, we examined the effects of CSP1–198, CSP1–112, and CSP1– 82 on the kinetics of guanine nucleotide exchange and GTP hydrolysis
We have found that CSP, a secretory vesicle protein, is able to significantly enhance the GTP hydrolysis of G␣s by stimulating GDP/GTP exchange

Summary

Introduction

G proteins constitute a family of heterotrimeric GTP-binding proteins that act as transducers in a variety of transmembrane signaling systems. Activation of G proteins involves an exchange of GDP for GTP on G␣ subunits and the release of GTP-bound G␣ and G␤␥ to interact with effector molecules. Our previous work has identified cysteine string protein (CSP) as a novel modulator of G proteins (1–3). The functional parallels between CSP and established G protein modulators are evident, the molecular mechanism by which CSP regulates G proteins is not yet known. CSPs are secretory vesicle proteins of 34 kDa that contain three conserved domains: a J domain, a linker domain, and a cysteine string region (Fig. 1A). CSP has been proposed to function as a trimeric complex with two molecular chaperones, Hsc (70-kDa heat shock cognate protein) (14 –16) and SGT (small glutamine-rich tetratricopeptide repeat domain protein) (17).

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