Characterization of the G alpha(s) regulator cysteine string protein.
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.
Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Michael
M
Natochin
Tessa N
TN
Campbell
Brandy
B
Barren
Linda C
LC
Miller
Shahid
S
Hameed
Nikolai O
NO
Artemyev
Janice E A
JE
Braun
D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D020558:GTP Phosphohydrolases / Q000737:chemistry; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D006147:Guanine / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D050883:HSC70 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D047468:Immunoprecipitation; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D018832:Molecular Chaperones / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors
D002352:Carrier Proteins; D018076:DNA, Complementary; D050883:HSC70 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; C494373:Hspa8 protein, rat; D008565:Membrane Proteins; D018832:Molecular Chaperones; D010455:Peptides; D011993:Recombinant Fusion Proteins; C114575:SGTA protein, human; C512694:Sgta protein, rat; C088460:cysteine string protein; D006147:Guanine; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D020558:GTP Phosphohydrolases; D019205:GTP-Binding Protein alpha Subunits, Gs; D002118:Calcium