Close association of the alpha subunits of Gq and G11 G proteins with actin filaments in WRK1 cells: relation to G protein-mediated phospholipase C activation.
A selective polyclonal antibody directed toward the C-terminal decapeptide common to the alpha subunits of Gq and G11 G proteins (G alpha q/G alpha 11) was prepared and used to investigate the subcellular distribution fo these proteins in WRK1 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.
Ibarrondo
J
J
Centre de Pharmacologie Endocrinologie, Institut National de la Santé et de la Recherche Médicale, Montpellier, France.
Joubert
D
D
Dufour
M N
MN
Cohen-Solal
A
A
Homburger
V
V
Jard
S
S
Guillon
G
G
D000199:Actins / Q000378:metabolism*; D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D003078:Colchicine / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects; D004789:Enzyme Activation; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007150:Immunohistochemistry; D007295:Inositol Phosphates / Q000096:biosynthesis; D051381:Rats; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*; D014667:Vasopressins / Q000494:pharmacology
D000199:Actins; D017607:Aluminum Compounds; D007295:Inositol Phosphates; D014667:Vasopressins; D005576:Colforsin; D015638:Cytochalasin D; D000242:Cyclic AMP; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D005459:Fluorides; D003078:Colchicine; C032311:aluminum fluoride