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Characterization of the G alpha(s) regulator cysteine string protein
Journal article   Open access   Peer reviewed

Characterization of the G alpha(s) regulator cysteine string protein

Michael Natochin, Tessa N Campbell, Brandy Barren, Linda C Miller, Shahid Hameed, Nikolai O Artemyev and Janice E A Braun
The Journal of biological chemistry, Vol.280(34), pp.30236-30241
08/26/2005
DOI: 10.1074/jbc.M500722200
PMID: 15972823
url
https://doi.org/10.1074/jbc.M500722200View
Published (Version of record) Open Access

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 CSP(1-198) (full-length), CSP(1-112), and CSP(1-82) on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with G alpha(s) and increases steady-state GTP hydrolysis. CSP(1-198) modulation of G alpha(s) was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP(1-112) was Hsc70-SGT-independent. CSP(1-112) preferentially associated with the inactive GDP-bound conformation of G alpha(s). Consistent with the stimulation of GTP hydrolysis, CSP(1-112) increased guanine nucleotide exchange of G alpha(s). The interaction of native G alpha(s) and CSP was confirmed by coimmunoprecipitation and showed that G alpha(s) associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the beta2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of G alpha(s) 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.
 GTP-Binding Protein alpha Subunits, Gs - metabolism Immunoprecipitation Molecular Chaperones - metabolism Calcium - metabolism Humans Immunoblotting HSP40 Heat-Shock Proteins HSC70 Heat-Shock Proteins Brain - metabolism Dose-Response Relationship, Drug GTP Phosphohydrolases - chemistry Time Factors Guanine - chemistry Guanosine Triphosphate - chemistry HSP70 Heat-Shock Proteins - chemistry Cyclic AMP - metabolism Protein Structure, Tertiary Cell Line Peptides - chemistry Signal Transduction Rats Recombinant Fusion Proteins - chemistry Hydrolysis Animals Carrier Proteins - metabolism Membrane Proteins - chemistry Protein Binding Protein Conformation Kinetics DNA, Complementary - metabolism

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