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A soluble C1b protein and its regulation of soluble type 7 adenylyl cyclase

Beeler, JA and Yan, SZ and Bykov, S and Murza, A and Asher, S and Tang, WJ (2004) A soluble C1b protein and its regulation of soluble type 7 adenylyl cyclase. Biochemistry, 43 (49). 15463 - 15471. ISSN 0006-2960

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Abstract

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 (Gsα) and with the conserved first catalytic domain (C1a) of type VII AC. We show that 7C1b-S inhibits Gsα-stimulated and Gsα-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.

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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Beeler, JA
Yan, SZ
Bykov, S
Murza, A
Asher, Sasher@pitt.eduASHER
Tang, WJ
Date: 14 December 2004
Date Type: Publication
Journal or Publication Title: Biochemistry
Volume: 43
Number: 49
Page Range: 15463 - 15471
DOI or Unique Handle: 10.1021/bi049088+
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Refereed: Yes
ISSN: 0006-2960
MeSH Headings: Adenylate Cyclase--antagonists & inhibitors; Adenylate Cyclase--chemistry; Adenylate Cyclase--isolation & purification; Adenylate Cyclase--metabolism; Amino Acid Sequence; Circular Dichroism; Conserved Sequence; Cytoplasm--enzymology; Cytoplasm--metabolism; Endopeptidase K--metabolism; Enzyme Activators--metabolism; GTP-Binding Protein alpha Subunits, Gs--antagonists & inhibitors; GTP-Binding Protein alpha Subunits, Gs--metabolism; Humans; Hydrolysis; Isoenzymes--antagonists & inhibitors; Isoenzymes--chemistry; Isoenzymes--isolation & purification; Isoenzymes--metabolism; Molecular Sequence Data; Peptide Fragments--antagonists & inhibitors; Peptide Fragments--chemistry; Peptide Fragments--isolation & purification; Peptide Fragments--metabolism; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits--antagonists & inhibitors; Protein Subunits--chemistry; Protein Subunits--isolation & purification; Protein Subunits--metabolism; Solubility; Spectrum Analysis, Raman
PubMed ID: 15581358
Date Deposited: 08 Feb 2013 21:03
Last Modified: 02 Feb 2019 16:55
URI: http://d-scholarship-dev.library.pitt.edu/id/eprint/17239

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