N-terminal Short Sequences of α Subunits of the G12Family Determine Selective Coupling to Receptors

Abstract

sequence homology of their ␣ subunits (1). The G 12 family is defined by G␣ 12 and G␣ 13 , and they share 67% amino acid sequence identity (2). Active forms of G␣ 12 and G␣ 13 induce several biological responses via activation of Rho, a major downstream common target for G␣ 12 and G␣ 13 , including stimulation of stress fiber formation and focal adhesion assembly (3), induction of neurite retraction (4), induction of apoptosis (5), transformation of fibroblasts (6), activation of phospholipase D (7), inhibition of Ca 2ϩ -dependent exocytosis (8), and activation of c-Jun N-terminal kinase (9). Recently, several proteins that interact with both G␣ 12 and G␣ 13 have been identified, including p115, a Rho guanine nucleotide exchange factor (10), cadherin (11), and radixin (12). Therefore, G␣ 12 and G␣ 13 are thought to transduce mutual downstream signaling through these effectors. In contrast, a variety of GPCRs appear to selectively couple to G␣ 12 or G␣ 13 (13). For example, thrombin and lysophosphatidic acid (LPA) have been shown to induce stress fiber formation via G␣ 12 and G␣ 13 , respectively, by using dominant negative mutants of G␣ 12 and G␣ 13 (13). However, this receptor-G protein coupling is not directly evaluated.Recently, we demonstrated that G␣ 12 and G␣ 13 interact with Ser/Thr phosphatase type 5 (PP5) through its tetratricopeptide repeat (TPR) domain and stimulate its phosphatase activity (14). In this study, we developed a novel assay to evaluate the activities of G␣ 12 and G␣ 13 by using glutathione S-transferase (GST)-fused TPR domain of PP5 (GST-TPR), taking advantage of the property that GST-TPR strongly interacts with active forms of G␣ 12 and G␣ 13 . By using this assay, we showed selective activations of G␣ 12 by thrombin and G␣ 13 by LPA, and found that N-terminal short sequences of G␣ 12 and G␣ 13 determine these selective activations. EXPERIMENTAL PROCEDURESMaterials-Agents obtained and commercial sources were as follows: thrombin and LPA, Sigma; rabbit polyclonal anti-G␣ 12 and anti-G␣ 13 antibodies and mouse monoclonal anti-RhoA antibody, Santa Cruz Biotechnology, Inc.; horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G antibody and horseradish peroxidase-conjugated swine anti-rabbit immunoglobulin G antibody, DAKO; and Chemiluminescence ECL Western blotting system, Amersham Biosciences. GSTfused proteins were purified from Escherichia coli as described previously (8,14). The TPR domain used for GST-TPR is a splicing variant of rat PP5 that contains residues 9 -122 following a new short coding region (12 amino acids; SRALGMGQLPAP) (14). The nucleotide sequence of this TPR variant is available from GenBank TM /EMBL/DDBJ under accession number AB101661.Plasmid Constructions-cDNAs of wild-type G␣ 12 and G␣ 13 (G␣ 12 WT and G␣ 13 WT), and their constitutively active mutants of G␣ 12 (G␣ 12 Q229L, G␣ 12 QL) and G␣ 13 (G␣ 13 Q226L, G␣ 13 QL) were obtained as described previously (4,14). To generate chimeric cDNA of G␣ 13 substituted with the N-terminal short sequence of G␣ 12 ...

Mitogenic signaling by lysophosphatidic acid (LPA) involves Gα12

“…While LPA readily activates Ga 12 in NIH3T3 cells, it has little or no effect on Ga 13 . In contrast, it appears that LPA stimulation is specific to Ga 13 in HEK293 cells (Yamaguchi et al, 2003;Ha and Dhanasekaran, unpublished results). Nonetheless, taken together with the previous studies that have shown that the transforming activity of Ga 13 is extremely weak compared to Ga 12 and the loss of Ga 13 does not affect the mitogenic response of Ga 13 À/À MEFs, our studies presented here point to a role for Ga 12 -and not to Ga 13 -in LPA-mediated mitogenic response.…”

“…Recently, it has been shown that the activated forms of Ga 12 and Ga 13 bind to the tetratricopeptide repeat (TPR) domain of Ser/Thr phosphatase type 5 (Yamaguchi et al, 2002(Yamaguchi et al, , 2003. Based on this observation, it has been demonstrated that the interaction between the glutathione S-transferase (GST)-fused TPR domain of PP5 (GST-TPR) and activated Ga 12 or Ga 13 can be used to determine the activation status of either of these asubunits (Yamaguchi et al, 2003).…”

“…Based on this observation, it has been demonstrated that the interaction between the glutathione S-transferase (GST)-fused TPR domain of PP5 (GST-TPR) and activated Ga 12 or Ga 13 can be used to determine the activation status of either of these asubunits (Yamaguchi et al, 2003). Therefore, to further validate the stimulation of Ga 12 by LPA in these cells, we examined the ability of GST-TPR to pull down Ga 12 from the lysates from LPA-stimulated (2 mM for 1 min) Ga 12 WT-NIH3T3 cells following the methods of Yamaguchi et al (2003). Lysates from Ga 12 WT-NIH3T3 cells exposed to 5% calf serum and Ga 12 QL-NIH3T3 To establish the functional coupling between LPAstimulated LPAR (LPA-LPAR) and Ga 12 , we decided to examine whether the stimulation of LPA can be correlated with a previously established Ga 12 -specific functional response.…”

“…values of three experiments and * denotes Po0.01 compared to unstimulated control value. (b) GST-TPR binding assay was carried out following the methods of Yamaguchi et al (2003). Ga 12 WT-NIH3T3, Ga 12 QL-NIH3T3, or pcDNA3-NIH3T3 were stimulated with either LPA (2 mM) or bovine serum (5%) for 1 min, rinsed twice with phosphate-buffered saline, and lysed with 500 ml of the ice-cold cell lysis buffer (25 mM HEPES, 0.1% Triton X-100, 300 mM NaCl, 20 mM b-glycerophosphate, 1.5 mM MgCl 2 , 0.2 mM EDTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM PMSF, 1 mM Na 3 VO 4 ).…”

Functional consequences of Gα13 mutations that disrupt interaction with p115RhoGEF

“…Next, we wanted to determine whether our mutants could interact with a 13 effectors other than p115Rho-GEF. It has been demonstrated that activated a 13 interacts with Ser/Thr protein phosphatase type 5 (PP5) through its TPR (Yamaguchi et al, 2002(Yamaguchi et al, , 2003. The purified TPR domain of PP5 fused to GST (GST-TPR) on glutathione agarose beads was incubated with COS-7 cell lysates expressing constitutively activated a 13 or mutants.…”