Direct genetic demonstration of G alpha 13 coupling to the orphan G protein-coupled receptor G2A leading to RhoA-dependent actin rearrangement - PubMed (original) (raw)

Direct genetic demonstration of G alpha 13 coupling to the orphan G protein-coupled receptor G2A leading to RhoA-dependent actin rearrangement

J H Kabarowski et al. Proc Natl Acad Sci U S A. 2000.

Abstract

G2A is an orphan G protein-coupled receptor (GPCR), expressed predominantly in T and B cells and homologous to a small group of GPCRs of unknown function expressed in lymphoid tissues. G2A is transcriptionally induced in response to diverse stimuli, and its ectopic expression suppresses transformation of B lymphoid precursors by BCR-ABL. G2A induces morphological transformation of NIH 3T3 fibroblasts. Microinjection of constructs encoding G2A into Swiss 3T3 fibroblasts induces actin reorganization into stress fibers that depends on RhoA, but not CDC42 or RAC. G2A elicits RhoA-dependent transcriptional activation of serum response factor. Direct evaluation of RhoA activity demonstrates elevated levels of RhoA-GTP in G2A-expressing cells. Microinjection of embryonic fibroblasts derived from various G alpha knockout mice establishes a requirement for G alpha 13 but not G alpha 12 or G alpha q/11 in G2A-induced actin rearrangement. In conclusion, G2A represents a family of GPCRs expressed in lymphocytes that may link diverse stimuli to cytoskeletal reorganization and transcriptional activation through a pathway involving G alpha 13 and RhoA.

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Figures

Figure 1

G2A induces morphological alterations in NIH 3T3 cells. (A) NIH 3T3 cells were infected with the indicated retroviruses and cultured for an additional 36 h. All populations were more than 90% GFP-positive by FACS analysis. (B) NIH 3T3 cells infected with the indicated retroviruses were cultured for 7 days.

Figure 2

(A) Microinjection of G2A induces stress fiber assembly in Swiss 3T3 cells. pEXV3 constructs encoding GFP, G2A.GFP, or G2A plus GFP were microinjected into nuclei of serum-starved Swiss 3T3 cells. Four hours later, cells were fixed and stained with rhodamine-conjugated phalloidin. In addition, pEXV3 G2A.GFP-injected cells were stained with a monoclonal antibody against Vinculin to visualize focal adhesion complexes. (B) Serum-starved Swiss 3T3 cells microinjected with a pEXV3 construct expressing V12 RAS were processed as in A. (Left) GFP fluorescence. (Right) Phalloidin-stained actin. Large arrows indicate injected cells. Small arrows in indicate lamellipodia in pEXV3 V12 RAS-injected cells.

Figure 3

Induction of stress fiber assembly by G2A is inhibited by N19 RhoA. Serum-starved Swiss 3T3 cells were microinjected with pEXV3 G2A and pEXV3 GFP plasmids together with pEXV3 constructs encoding (A) MYC epitope-tagged N19 RhoA, (B) MYC epitope-tagged N17 CDC42, or (C) MYC epitope-tagged N17 Rac1. Cells were fixed 4 h later and stained with 9E10 mAb to detect expression of dominant negative Rho family mutants (Middle) and rhodamine-conjugated phalloidin (Bottom). Large arrows indicate injected cells. (Bar = 50 μm.)

Figure 4

(A) G2A induces transcriptional activation of SRF. NIH 3T3 cells were cotransfected with reporter plasmid pSRF-Luc, pTK-Renilla Luc, and pEXV3 G2A or pEXV3 GFP with or without pRK5 mycC3T (totaling 0.9 μg DNA). Twenty four hours after transfection, cells were lysed and subjected to luciferase assays. Transfections and SRF-Luc assays were performed in triplicate, and results presented are typical of three independent experiments. (B) G2A activates RhoA in Swiss 3T3 cells. Swiss 3T3 cells were infected with MSCV GFP or MSCV G2AiresGFP retroviruses and 24 h later were serum-starved for 12 h. Cells subsequently were lysed, and lysates were incubated on ice for 1 h with glutathione _S_-transferase-RRBD immobilized on glutathione-agarose beads to affinity precipitate RhoA-GTP. Affinity precipitates (RhoA GTP) and aliquots of total lysates (total RhoA) were Western blotted with a mAb against RhoA. Lanes 2 and 4, lysophosphatidic acid (LPA) (1 μg/ml, 5 min) stimulated activation of RhoA. Lanes 1 and 3, Increased levels of RhoA GTP in G2A expressing Swiss 3T3 cells compared with control Swiss 3T3 cells.

Figure 5

G2A-induced stress fiber assembly requires Gα13. The indicated MEFs were microinjected with pEXV3 G2A.GFP, fixed 4 h later, and stained with rhodamine-conjugated phalloidin. (Upper) GFP fluorescence. (Lower) Phalloidin-stained actin. Arrows indicate injected cells. (Bar = 50 μm.)

Figure 6

Reconstitution of functional Gα13 in Gα13 KO MEFs restores their responsiveness to G2A. Gα13 KO MEFs were microinjected with pEXV3 GFP or pEXV3 G2A.GFP together with pCIS Gα13, fixed 4 h later, and stained with rhodamine-conjugated phalloidin (Bottom), and rabbit antiserum against Gα13 to detect ectopically expressed Gα13 (Middle). (Upper) GFP fluorescence. Arrows indicate injected cells. (Bar = 20 μm.)

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Direct genetic demonstration of G alpha 13 coupling to the orphan G protein-coupled receptor G2A leading to RhoA-dependent actin rearrangement - PubMed (original) (raw)