IMMUNOCHEMICAL IDENTIFICATION AND DIFFERENTIAL PHOSPHORYLATION OF ALTERNATIVELY SPLICED FORMS OF THE ALPHA(1A) SUBUNIT OF BRAIN CALCIUM CHANNELS

被引:40
作者
SAKURAI, T
HELL, JW
WOPPMANN, A
MILJANICH, GP
CATTERALL, WA
机构
[1] UNIV WASHINGTON,DEPT PHARMACOL,SEATTLE,WA 98195
[2] NEUREX CORP,MENLO PK,CA 94025
关键词
D O I
10.1074/jbc.270.36.21234
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Biochemical properties of the alpha(1) subunits of class A brain calcium channels (alpha(1A)) were examined in adult rat brain membrane fractions using a site-directed antipeptide antibody (anti-CNA3) specific for alpha(1A). Anti-CNA3 specifically immunoprecipitated high affinity receptor sites for omega-conotoxin MVIIC (K-d similar to 100 pM), but not receptor sites for the dihydropyridine isradipine or for omega-conotoxin GVIA. In immunoblotting and immunoprecipitation experiments, anti-CNA3 recognized at least two distinct immunoreactive alpha(1A) polypeptides, a major form with an apparent molecular mass of 190 kDa and a minor, full-length form with an apparent molecular mass of 220 kDa. The 220- and 190-kDa alpha(1A) polypeptides were also specifically recognized by both anti-BI-Nt and anti-HI-1-Ct antibodies, which are directed against the NH2- and COOH-terminal ends of alpha(1A) predicted from cDNA sequence, respectively. These data indicate that the predicted NH2 and COOH termini are present in both size forms and therefore that these isoforms of alpha(1A) are created by alternative RNA splicing rather than post-translational proteolytic processing of the NH2 or COOH termini. The 220-kDa form was phosphorylated preferentially by cAMP-dependent protein kinase, whereas protein kinase C and cGMP-dependent protein kinase preferentially phosphorylated the 190-kDa form. Our results identify at least two distinct alpha(1A) subunits with different molecular mass, demonstrate that they may result from alternative mRNA splicing, and suggest that they may be differentially regulated by protein phosphorylation.
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页码:21234 / 21242
页数:9
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