A soluble C1b protein and its regulation of soluble type 7 adenylyl cyclase.
Adenylyl cyclase (AC) is a prototypical cell-signaling molecule expressed in virtually all organisms from bacteria to man. While C1b, a poorly conserved region within mammalian AC, has been implicated in numerous isoform-specific regulatory properties, no one has purified the C1b region as a functional protein to homogeneity in order to study its role in enzyme function. We hypothesize that C1b is an internal regulatory subunit. To pursue this hypothesis, we constructed several soluble C1b proteins from type VII AC, arriving at one, 7C1b-S, which can be expressed and purified from Escherichia coli. 7C1b-S is relatively stable, as demonstrated by limited proteolytic analysis, circular dichroism, and UV Raman spectroscopy. Using size-exclusion chromatography and co-immunoprecipitation we demonstrate that 7C1b-S interacts with a cardinal activator of AC (Gsalpha) and with the conserved first catalytic domain (C1a) of type VII AC. We show that 7C1b-S inhibits Gsalpha-stimulated and Gsalpha-forskolin stimulated activity in our soluble ACVII model system. On the basis of these results, we suggest that 7C1b-S meets basic criteria to serve as a model protein for the C1b region and may be used as a prototype to develop other isoform C1b soluble model proteins to further investigate the role of this domain in isoform-specific regulation of adenylyl cyclase.
Beeler
Jeff A
JA
Ben May Cancer Research Institute and Committee on Neurobiology, University of Chicago, Chicago, Illinois 60637, USA.
Yan
Shui-Zhong
SZ
Bykov
Sergei
S
Murza
Adrian
A
Asher
Sanford
S
Tang
Wei-Jen
WJ
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D017124:Conserved Sequence; D003593:Cytoplasm / Q000201:enzymology* / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D020536:Enzyme Activators / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013059:Spectrum Analysis, Raman
D000067956:Adenylyl Cyclase Inhibitors; D020536:Enzyme Activators; D007527:Isoenzymes; D010446:Peptide Fragments; D021122:Protein Subunits; D019286:Endopeptidase K; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; C473265:adenylyl cyclase 7