No abstract

Despite advances in the understanding of pathways regulated by the G 12 family of heterotrimeric G proteins, much regarding the engagement of this family by receptors remains unclear. We explore here, using the thromboxane A 2 receptor TP␣, the ability of G 12 and G 13 to report differences in the potency and efficacy of receptor ligands. We were interested especially in the potential of the isoprostane 8-iso-prostaglandin F 2␣ (8-iso-PGF 2␣ ), among other ligands examined, to activate G 12 and G 13 through TP␣ explicitly. We were also interested in the functionality of TP␣-G␣ fusion proteins germane to G 12 and G 13 . Using fusion proteins in Spodoptera frugiperda (Sf9) cells and independently expressed proteins in human embryonic kidney 293 cells, and using guanosine 5Ј-O-(3-[35 S]thio)triphosphate binding to evaluate G␣ activation directly, we found for G␣ 13 that no ligand tested, including 8-iso-prostaglandin F 2␣ (8-iso-PGF 2␣ ) and a purported antagonist (pinane thromboxane A 2 ), was silent. The activity of agonists was especially pronounced when evaluated for TP␣-G␣ 13 and in the context of receptor reserve. Agonist activity for 8-iso-PGF 2␣ was diminished and that for pinane thromboxane A 2 nonexistent when G␣ 12 was the reporter. These data establish that G 12 and G 13 can report differentially potency and efficacy and underscore the relevance of receptor and G protein context.The G 12 family of heterotrimeric G proteins, comprising G 12 and G 13 in vertebrates, has received considerable attention for its roles in cell contractility, motility, and proliferation (for review, see Riobo and Manning, 2005). Specific functions for this family have been deduced through the actions of constitutively active G␣ subunits, effects of deleting one or both G␣ genes, effects of dominant-negative molecules, and interactions of the G␣ subunits with other proteins. Despite advances in understanding the pathways regulated by the G 12 family, however, much regarding the engagement of this family by agonists remains unclear. At a basic level, the extent to which G 12 and G 13 can report differences among agonists, for example, in terms of potency and efficacy, is entirely unknown, nor can it be easily predicted given the unusual properties of the respective G␣ subunits (Singer et al., 1994;Kozasa and Gilman, 1995). One of the problems in evaluating the dynamics of signaling through G 12 and G 13 is that enzymes or ion channels uniquely regulated by the two proteins and whose activities are easily measured have not yet been identified. A related problem is that receptors having the capacity to couple to G 12 and/or G 13 invariably couple to other G proteins as well. The analysis of proximal signaling so important to the modeling of receptor function in the context of other G proteins, therefore, has proven difficult for the G 12 family and, except for measurements of frank activation, has not been approached.Thromboxane A 2 is a member of the prostaglandin family of lipid mediators generated after cyclooxygenase-c...

Regions of basic amino acids in proteins can promote membrane localization through electrostatic interactions with negatively charged membrane lipid head groups. Previous work showed that the heterotrimeric G protein subunit ␣ q contains a polybasic region in its N terminus that contributes to plasma membrane localization. Here, the role of the N-terminal polybasic region of ␣ q in signaling was addressed. For ␣ q mutants, loss of plasma membrane localization correlated with loss of signaling function, as measured by the ability to couple activated G protein-coupled receptors (GPCRs) to stimulation of inositol phosphate production. However, recovery of plasma membrane localization of ␣ q polybasic mutants by introduction of a site for myristoylation or by coexpression of ␤␥ failed to recover signaling, suggesting a role for N-terminal basic amino acids of ␣ q beyond simple plasma membrane localization. It is noteworthy that an ␣ q 4Q mutant, containing glutamine substitutions at arginines 27, 30, 31, and 34, was identified that failed to mediate signaling yet retained plasma membrane localization. Although ␣ q 4Q failed to couple activated receptors to inositol phosphate production, it was able to bind ␤␥, bind RGS4 in an activation-dependent manner, stimulate inositol phosphate production in a receptor-independent manner, and productively interact with a GPCR in isolated membranes. It is noteworthy that ␣ q 4Q showed a differing localization to plasma membrane nanodomains compared with wild-type ␣ q . Thus, basic amino acids in the N terminus of ␣ q can affect its lateral segregation on plasma membranes, and changes in such lateral segregation may be responsible for the observed signaling defects of ␣ q 4Q.