Leukemia-associated Rho guanine nucleotide exchange factor promotes G alpha q-coupled activation of RhoA

Abstract

Leukemia-associated Rho guanine-nucleotide exchange factor (LARG) belongs to the subfamily of Dbl homology RhoGEF proteins (including p115 RhoGEF and PDZ-RhoGEF) that possess amino-terminal regulator of G protein signaling (RGS) boxes also found within GTPase-accelerating proteins (GAPs) for heterotrimeric G protein alpha subunits. p115 RhoGEF stimulates the intrinsic GTP hydrolysis activity of G alpha 12/13 subunits and acts as an effector for G13-coupled receptors by linking receptor activation to RhoA activation. The presence of RGS box and Dbl homology domains within LARG suggests this protein may also function as a GAP toward specific G alpha subunits and couple G alpha activation to RhoA-mediating signaling pathways. Unlike the RGS box of p115 RhoGEF, the RGS box of LARG interacts not only with G alpha 12 and G alpha 13 but also with G alpha q. In cellular coimmunoprecipitation studies, the LARG RGS box formed stable complexes with the transition state mimetic forms of G alpha q, G alpha 12, and G alpha 13. Expression of the LARG RGS box diminished the transforming activity of oncogenic G protein-coupled receptors (Mas, G2A, and m1-muscarinic cholinergic) coupled to G alpha q and G alpha 13. Activated G alpha q, as well as G alpha 12 and G alpha 13, cooperated with LARG and caused synergistic activation of RhoA, suggesting that all three G alpha subunits stimulate LARG-mediated activation of RhoA. Our findings suggest that the RhoA exchange factor LARG, unlike the related p115 RhoGEF and PDZ-RhoGEF proteins, can serve as an effector for Gq-coupled receptors, mediating their functional linkage to RhoA-dependent signaling pathways.

FIG. 1.

Functional domains and structural mutants of LARG. Schematic representations of full-length (FL), amino-terminal truncation mutants (ΔN308 and ΔN754), and the isolated RGS box (LARG-box) proteins are shown. Numbers correspond to amino acids. HA, HA epitope tag; PDZ, PSD-95/Discs-large/ZO-1 domain; NLS, putative nuclear localization signal; DH, DH domain; PH, PH domain.

FIG. 2.

LARG RGS box complexes with activated Gα12, Gα13, and Gαq subunits in vivo. NIH 3T3 cells were cotransfected with vectors that express HA epitope-tagged versions of the isolated RGS box of p115 RhoGEF or LARG (1 μg) and expression vectors encoding wt Gα12, Gα13, or Gαq (1 μg). Whole-cell lysates were prepared in the presence or absence of AlF₄⁻, which activates Gα subunits to adopt a conformation mimicking the transition-state for GTP hydrolysis (44). HA-tagged RGS box proteins were immunoprecipitated (IP) from whole cell lysates (800 μg of total protein) with an anti-HA (αHA) monoclonal antibody and resolved by SDS-PAGE. Immunoprecipitated p115-RGS and LARG-RGS protein and coimmunoprecipitated Gα12, Gα13, and Gαq subunits were detected by immunoblot analyses (IB). To control for expression levels, separate aliquots of each lysate (20 μg) were taken before immunoprecipitation and resolved by SDS-PAGE and amounts of Gα12, Gα13, or Gαq were detected by immunoblot analyses.

FIG. 3.

LARG RGS box attenuates Gα12-, Gα13-, and Gαq-mediated SRF activation in NIH 3T3 cells. NIH 3T3 cells were transiently cotransfected with expression plasmids (1 μg) encoding LARG RGS, p115 RGS, RGS2, and GTPase-deficient Gα12 (Q229L), Gα13 (Q226L), or Gαq (Q229L) proteins and a luciferase reporter plasmid (0.5 μg) that is responsive to activation of SRF [(SREm)2-Luc]. After transfection, cells were placed in 0.1% calf serum for 24 h. Cell lysates were then analyzed for luciferase activity and normalized to cells transfected with empty vectors (fold activation). All reporter assays were performed in six-well plates in duplicate. Total cell lysates (30 μg) were analyzed for protein expression by Western blot analysis with anti-Gα12, -Gα13, -Gαq, or -HA antibodies (data not shown). The data shown represent the averages of three experiments (± standard errors of the means).

FIG. 4.

LARG RGS box attenuates agonist-dependent m1-mAChR-mediated SRF activation in NIH 3T3 cells. NIH 3T3 cell lines stably expressing the Gq-coupled m1-mAChR and the indicated RGS box proteins were transiently transfected with the (SREm)2-Luc SRF-responsive reporter plasmid (0.5 μg). NIH 3T3 cells stably expressing m1-mAChR alone were supertransfected with (SREm)2-Luc (0.5 μg) and RGS2 (1 μg). After transfection, cells were placed in growth medium supplemented with 0.1% calf serum for 24 h. Cell lysates were than analyzed for luciferase activity, and fold activation was determined by the number of relative luciferase units relative to the number of units seen with the empty vector control. All reporter assays were performed in six-well plates in duplicate. The data shown are representative of at least three independent assays performed on duplicate plates.

FIG. 5.

LARG RGS box abolishes focus formation mediated by Gαq- and Gα13-coupled GPCRs. (A) LARG RGS box blocks G2A-mediated transformation of NIH 3T3 cells. G2A is an oncogenic GPCR that couples to Gα13 (20). NIH 3T3 cells were cotransfected with expression vectors encoding transforming oncoproteins G2A (0.5 μg) or activated HRas(61L) (25 ng) along with vectors (1 μg) that express the RGS box of p115 RhoGEF (p115-RGS) or LARG (LARG-RGS). Focus-forming activity was determined 14 days after transfection. The data shown represent the average number of foci of three experiments performed in duplicate. (B) LARG RGS box blocks Mas-mediated transformation of NIH 3T3 cells. Mas is an oncogenic GPCR that couples to Gαq and Gαi. NIH 3T3 cells were cotransfected with plasmid vectors encoding Mas (0.5 μg) or HRas(61L) (25 ng) either alone or together with vectors (1 μg) encoding p115-RGS or LARG-RGS. Focus-forming activity was determined 14 days after transfection. The data shown represent the average number of foci of three experiments performed in duplicate. (C) LARG RGS box blocks agonist-dependent m1-mAChR-mediated transformation of NIH 3T3 cells. Stable NIH 3T3 cell lines were established expressing the Gq-coupled m1-mAChR and the indicated RGS box proteins. Cells were maintained in growth medium supplemented with either 100 μM carbachol (an m1-mAChR agonist) or vehicle, and focus-forming activity was determined after 12 days. The data shown represent the average number of foci of three individual experiments performed in duplicate.

FIG. 6.

Coexpression of activated Gα12, Gα13, and Gαq enhances LARG-mediated RhoA activation. NIH 3T3 cells were transiently transfected with expression plasmids (1 μg) encoding activated mutants of Gα12 (A), Gα13 (B), Gαq (C); full-length (2 μg), ΔN308, or ΔN754 LARG; or the indicated combinations of activated Gα subunits and LARG. After transfection, cells were cultured for 24 h in growth medium supplemented with low serum (0.1%) and lysed, and the lysates (800 μg total protein) were used in GST pull-down assays using GST-rhotekin RBD immobilized on glutathione-agarose beads (20 μg). Bound proteins and total cell lysates were analyzed by Western blot analyses with anti-RhoA antibodies. The expression of Gα subunits and LARG was analyzed by Western blot analyses of total cell lysates using antibodies to Gα12, Gα13, Gαq, or HA (data not shown).