Our results clearly show that a deletion of Gαi3 augments Gαi2-mediated migratory responses toward CXCR3 ligands, whereas deletion of Gαi2 ablates the response. The Gαi3-mediated inhibition of CXCR3 signaling is not reliant on the exchange of GDP for GTP by Gαi3, but it does require ligand-stimulated interaction between Gαi3 and the CXCR3 receptor. Presumably, a conformational change of the receptor caused by ligand binding provides conditions that are optimal for Gαi3 binding to the receptor. However, the receptor-bound Gαi3 appears unable to undergo a conformational change required for dissociation of GDP and/or binding to GTP, thus preventing it from full activation and leaving the receptor. By occupancy of the receptor, Gαi3 blocks Gαi2 activation. It has been shown that the DRY motif at the N-terminus of the second intracellular loop is key for proper G protein recognition and interaction (42
). Our data suggest that this motif may be also essential for the blockade induced by Gαi3, which requires further investigation to be concluded. Substitution of arginine (R)149
amino residue to asparagine (N) ablates not only activation of Gαi2 but also binding of Gαi3 to the receptor. Under similar conditions, receptors carrying mutations at the C-terminus can activate Gαi2 and preserve Gαi3 interaction similarly to the WT CXCR3 receptor (). Lack of Gαi3 binding to and Gαi2 activation by the R149
N mutant cannot be ascribed to the levels of receptor expression on the cells. Although the R149
N-CXCR3 mutant was expressed on the cells at levels slightly lower than the WT CXCR3 receptor, its expression was comparable to the LLL-CXCR3 mutant () (24
). The latter could activate Gαi2 and bind with Gαi3 as the WT CXCR3 receptor (). Murine Gαi2 and Gαi3 are 83% identical in amino residue sequence. This structural similarity may be the basis for their competition for the same binding domain of the CXCR3 receptor or for Gαi3 interference with Gαi2 interaction of the receptor. A subtle difference in the receptor conformation induced by a specific agonist is likely to determine whether the activated receptor can interact and activate a given Gαi protein or interact with a Gαi protein without inducing its full activation. Therefore, function of a given Gαi protein as an activator or inhibitor is receptor specific and the interplay between these two G proteins may be essential in the control of the amplitude of a GPCR-induced signal. To the best of our knowledge, this is the first description of a G protein interacting with a receptor after ligand binding for the sole purpose of diminishing the other G protein interaction with the receptor. This finding gives novel insight into regulation of GPCRs’ signaling by heterotrimeric G proteins.
GPCRs initiate a signaling cascade through activation of heterotrimeric G proteins following stimulation by an extracellular agonist (43
). To meet the complexity and versatility of cell migration in the body, many chemokine receptors have evolved to recognize more than one chemokine, such as the CXCR3 and CCR7 receptors, or one chemokine receptor can be activated by multiple chemokines like CCL5 (RANTES), CCL3 (MIP-1α), MCP-2 and MCP-3 (44
). Our finding that a migratory response is tied proportionally to the amplitude of G protein activation indicates that stoichiometry of G protein activation is directly linked to the propensity of a cell to migrate. The precise activation level of the Gαi2 protein stimulated by ligation of the CXCR3 receptor is determined by the affinity of the ligand as well as the interplay of Gαi2 and Gαi3 with the receptor. In accordance with CXCR3 signaling dependent of Gαi2 and inhibition of the signaling by Gαi3, our in vivo study showed that similar to T cells from mice lacking the CXCR3 receptor, Gαi2-deficient T cells failed to elicit an acute graft-vs.-host defense (GVHD) reaction after transfused into full MHC-mismatched Balb/c SB-17 SCID (severe combined immunodeficiency) mice (Jin et al
., manuscript in preparation) (30
). In contrast, transfer of Gαi3-deficient T cells into the SCID mice stimulated an aggravating GVHD response compared to WT T cells. Thus, an interplay among different heterotrimeric G proteins is likely to have a role to play in vivo, introducing another level of complexity in the control of cell migration systemically. The complex regulation of GPCR signaling at multiple levels is crucial in temporal and spatial regulation of cell migration in the body.
Determination of receptors linked to Gαi proteins has been greatly aided by the use of PTX. PTX has also allowed insight into downstream effectors that are either partially or fully involved in propagating a signal by Gαi proteins. While PTX has provided extensive and important details about the agonist and signal transduction mechanisms, the specifics about individual Gαi/o family members has gone largely unresolved because PTX blocks receptor interaction by several Gαi/o family members. With the advent of the genetic manipulation of mice, we can tease out the specifics of Gαi1, Gαi2, and Gαi3 in a migratory response stimulated by a specific receptor. Studies of B cell migration in Gαi2-/- mice suggest an irreplaceable role of Gαi2 in chemotactic responses to chemokines CXCL12 and CXCL13 (14
). On the other hand, Gαi2 and Gαi3 are redundant in thymic emigration as indicated by no significant defects in thymic exportation in Gαi2- and Gαi3-deficient mice (12
). Although the proportions and the numbers of CD4 and CD8 single positive thymocytes were increased in Gαi2-/- mice as lck
-PTX-transgenic mice, the increases were not caused by a defect in thymic egress, rather by an accelerated transition from the double positive to single positive thymocytes as shown by our previous investigation (12
). In support, nearly normal numbers and percentages of CD4+ and CD8+ T cells were seen in the spleen of Gαi2-/- mice (11
). Likewise, mature thymocytes in Gαi3-/- mice populated lymphoid tissues normally in the periphery and there was no aberrant accumulation of single positive thymocytes in the mice (13
). The redundancy of Gαi2 and Gαi3 in thymic egress was also consistent with our recent study showing that Gαi2-KO and Gαi3-KO T cells migrated indistinguishably from WT T cells in response to an increasing concentration of sphingosine-1-phosphate (S1P), a lipid mediator controlling T cell egress (our unpublished data) (47
How the redundancy works mechanistically is not fully understood at present. Association with the same subunit of Gβγ may be one of the common mechanisms whereby Gαi2 and Gαi3 can compensate for the absence of one to another. Hwang et al. have shown that functional silence of either Gαi2 or Gαi3 by specific siRNA has little effect on the migration of macrophages toward an increasing concentration gradient of C5a or C3a (48
). Deletion of Gβ2, however, ablated C5a- or C3a-provoked migration, suggesting that both Gαi2 and Gαi3 can transduce the migration signal as long as the Gβ2 subunit is presented (48
). On the contrary, the CXCR4 receptor requires both Gαi2 and Gαi3 for a full response. Absence of either gene impaired T cell migration induced by CXCL12 (), although decreased expression of the CXCR4 receptor on these cells may also be partially involved. We should point out that a slight increase or decrease (10~15%) in the Gαi2 expression levels in Gαi3-/- T cells or Gαi3 in Gαi2-/- T cells was observed in some mice (). However, the levels of Gαi protein expression were not correlated with migratory changes for any of the studied chemokines. In particular, the slight decrease in Gαi2 expression in Gαi3-KO T cells was discordant with the increased activity for the CXCR3 receptor. Similarly, although the levels of Gαi2 and Gαi3 expression were slightly decreased in activated as compared to non-activated T cells, the decrease affects little their chemotactic response to CCL19 stimulation (). This is probably due to the following two reasons. First, Gαi2 or Gαi3 is a lot more abundant than any individual chemokine receptor in a cell. Secondly, constant recycling of Gαi proteins from an active to inactive status further increases the size of a Gαi protein pool that is already far in excess of any individual chemokine receptor. Therefore, distinct function of Gαi2 and Gαi3 described in this study is unlikely due to a slightly altered level of Gαi2 or Gαi3 protein. Nevertheless, this does not exclude that the absolute levels of Gαi2 and Gαi3 can affect activation of a given GPCR, when the relative levels of Gαi2 and Gαi3 differ drastically. Our data thus argue that Gαi2 and Gαi3 proteins may play overlapping, distinct, antagonizing and additive roles depending on a specific receptor. The Gαi2 and Gαi3-null mutation mice provide a unique system to explore fundamental signaling differences of a specific GPCR coupling to Gαi2, Gαi3 or both and potential interplay between these Gαi proteins.