N-terminal short sequences of alpha subunits of the G12 family determine selective coupling to receptors.

The Galpha subunits of the G(12) family of heterotrimeric G proteins, defined by Galpha(12) and Galpha(13), have many cellular functions in common, such as stress fiber formation and neurite retraction. However, a variety of G protein-coupled receptors appear to couple selectively to Galpha(12) and Galpha(13). For example, thrombin and lysophosphatidic acid (LPA) have been shown to induce stress fiber formation via Galpha(12) and Galpha(13), respectively. We recently showed that active forms of Galpha(12) and Galpha(13) interact with Ser/Thr phosphatase type 5 through its tetratricopeptide repeat domain. Here we developed a novel assay to measure the activities of Galpha(12) and Galpha(13) by using glutathione S-transferase-fused tetratricopeptide repeat domain of Ser/Thr phosphatase type 5, taking advantage of the property that tetratricopeptide repeat domain strongly interacts with active forms of Galpha(12) and Galpha(13). By using this assay, we identified that thrombin and LPA selectively activate Galpha(12) and Galpha(13), respectively. Galpha(12) and Galpha(13) show a high amino acid sequence homology except for their N-terminal short sequences. Then we generated chimeric G proteins Galpha(12N/13C) and Galpha(13N/12C), in which the N-terminal short sequences are replaced by each other, and showed that thrombin and LPA selectively activate Galpha(12N/13C) and Galpha(13N/12C), respectively. Moreover, thrombin and LPA stimulate RhoA activity through Galpha(12) and Galpha(13), respectively, in a Galpha(12) family N-terminal sequence-dependent manner. Thus, N-terminal short sequences of the G(12) family determine the selective couplings of thrombin and LPA receptors to the Galpha(12) family.