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

A selective polyclonal antibody directed toward the C-terminal decapep tide 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 di stribution 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 thes e alpha subunits. Functional studies indicated that this antibody inhi bited vasopressin- and guanosine 5'-[alpha-thio] triphosphate-sensitiv e phospholipase C activities. Immunofluorescence experiments done with this antibody revealed a filamentous labeling corresponding to intrac ytoplasmic and perimembranous actin-like filament structures. Colocali zation of G(alpha q)/G(alpha 11) and F-actin filaments (F-actin) was d emonstrated by double-labeling experiments with anti-G(alpha q)/G(alph a 11) and anti-actin antibodies. Immunoblot analysis of membrane, cyto skeletal, and F-actin-rich fractions confirmed the close association o f G(alpha q)/G(alpha 11) with actin, Large amounts of G(alpha q)/G(alp ha 11) were recovered in the desmin- and tubulin-free F-actin-rich fra ction obtained by a double depolymerization-repolymerization cycle. Di sorganization of F-actin filaments with cytochalasin D preserved G(alp ha q)/G(alpha 11) and F-actin colocalization but partially inhibited v asopressin- and fluoroaluminate-sensitive phospholipase C activity, su ggesting that actin-associated G(alpha q)/G(alpha 11) proteins play a role in signal transduction.