CLOSE ASSOCIATION OF THE ALPHA-SUBUNITS OF G(Q) AND G(11) G-PROTEINS WITH ACTIN-FILAMENTS IN WRK(1) CELLS - RELATION TO G PROTEIN-MEDIATED PHOSPHOLIPASE-C ACTIVATION

被引:64
作者
IBARRONDO, J
JOUBERT, D
DUFOUR, MN
COHENSOLAL, A
HOMBURGER, V
JARD, S
GUILLON, G
机构
[1] INSERM,U401,CTR PHARMACOL ENDOCRINOL,F-34094 MONTPELLIER 05,FRANCE
[2] CTR PHARMACOL ENDOCRINOL,CNRS,U9023,F-34094 MONTPELLIER 05,FRANCE
关键词
CYTOSKELETON;
D O I
10.1073/pnas.92.18.8413
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
A selective polyclonal antibody directed toward the C-terminal decapeptide common to the cu subunits of G(q) and G(11) G proteins (G(alpha q)/G(alpha 11)) was prepared and used to investigate the subcellular distribution of these proteins in WRK(1) cells, a rat mammary tumor cell line. In immunoblots, the antibody recognized purified G(alpha q) and G(alpha 11) proteins and labeled only two bands corresponding to these alpha subunits. Functional studies indicated that this antibody inhibited vasopressin- and guanosine 5'-[alpha-thio] triphosphate-sensitive phospholipase C activities. Immunofluorescence experiments done with this antibody revealed a filamentous labeling corresponding to intracytoplasmic and perimembranous actin-like filament structures. Colocalization of G(alpha q)/G(alpha 11) and F-actin filaments (F-actin) was demonstrated by double-labeling experiments with anti-G(alpha q)/G(alpha 11) and anti-actin antibodies. Immunoblot analysis of membrane, cytoskeletal, and F-actin-rich fractions confirmed the close association of G(alpha q)/G(alpha 11) with actin, Large amounts of G(alpha q)/G(alpha 11) were recovered in the desmin- and tubulin-free F-actin-rich fraction obtained by a double depolymerization-repolymerization cycle. Disorganization of F-actin filaments with cytochalasin D preserved G(alpha q)/G(alpha 11) and F-actin colocalization but partially inhibited vasopressin- and fluoroaluminate-sensitive phospholipase C activity, suggesting that actin-associated G(alpha q)/G(alpha 11) proteins play a role in signal transduction.
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页码:8413 / 8417
页数:5
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