Although G protein coupled receptors (GPCRs) are often pictured as acting through specific, dedicated G proteins, it is now known that a single GPCR can bind and activate G proteins from more than one G alpha class [see 26]
. PAR1, a thrombin receptor, can bind to Gαi/o
, or to Gα12/13 
. β2-adrenergic receptors, when phosphorylated by PKA, switch affinities from Gαs
to Gαi 
. The class I metabotropic glutamate receptor mGluR1 has been shown to bind Gαi/o
, and Gαs
, at least in certain cell types 
. Wang et al. 
found that parathyroid hormone receptor 1 regulates different genes with different G proteins or combinations of G proteins, suggesting that individual G proteins might be required for some cell behaviors but not for others. These are just a few of the many examples of GPCRs coupling to multiple G proteins.
Chemokine receptors as a class are generally thought to signal through Gαi/o
-type G proteins to decrease cAMP, activate PI3K, and activate both p38 and ERK1/2 MAP Kinases (see 
for reviews). PI3K activation leads to activation of a number of other kinases, including Akt. SDF1 signaling through the chemokine receptor CXCR4 is also associated with changes in transcription, usually mediated through MAPK or Akt, that contribute to cell survival 
. However, several groups have found that CXCR4 signals through other classes of G proteins. Maghazachi 
reported that antibodies targeting Gαo
, but not Gαi
, or Gαz
, could block SDF1-induced chemotaxis in natural killer cells. Soede et al. 
found that CXCR4-dependent migration of myeloid leukemia cells require either the combination of Gαi and Gαq
alone, depending on the destination tissue. Tan et al. 
showed that SDF1/CXCR4-induced migration of Jurkat T cells required both Gα13
, which activated Rho, and Gαi
. These and other studies raised the possibility that SDF1/CXCR4 signaling in axon guidance might be more complex than that of the classic chemokine signaling pathway. Previous work from our laboratory 
identified several components of SDF1/CXCR4 signaling in the antirepellent pathway, including a pertussis toxin-sensitive G protein, increased cAMP, and activation of PKA. In addition to the surprising apparent increase in cAMP levels observed in these previous studies, the effects of SDF1 on axonal responses to repellents were found to be independent of PI3K/Akt signaling and of MAPK.
The findings in this study show that SDF1's antirepellent activity can be blocked separately by Gαi, Gαq/11, or Gβγspecific competitive inhibitors. These data suggest that each is required for the normal function of the antirepellent pathway. However, we also found that overexpression of constitutively active forms of Gαi or of Gαq can mimic application of SDF1. This suggests that either one of these signaling components is capable of stimulating a common downstream element that is sufficient for activation of the pathway. These findings are consistent with the idea that SDF1 stimulates multiple G protein coupled pathways to a degree that is insufficient for any one of them alone to induce a physiological response, but in combination, their actions sum to a level above a threshold for activation to produce an antirepellent response.
We also found that overexpression of a constitutively active Gαs can mimic SDF1 even though a competitive inhibitor of Gαs does not block SDF1 mediated signaling. As Gαs is a canonical stimulator of adenylate cyclase activity and would be expected to elevate cAMP levels, this finding is consistent with the idea that the common element upon which Gαi, Gαq, and Gβγ all converge downstream from SDF1 activation of CXCR4 is elevated cAMP levels. Thus, our proposed model of the signaling pathway is that Gαi, Gαq, and their associated βγ subunits all cooperate to increase the local concentration of cAMP, leading to suppression of axonal repulsion (). The ability of Gαs to accomplish the same thing through a different route raises the possibility that a very wide range of GPCRs could influence axonal responses to repellents and axonal pathfinding.
A model for antirepellent signaling.
Previous work has shown that SDF1's antirepellent activity requires calmodulin and the calcium/calmodulin-stimulated cyclase ADCY8 
. Xu 
also showed by Förster resonance energy transfer (FRET) that SDF1 stimulates increased cAMP levels, and that this can be blocked by inhibition of calmodulin. Gαi
are not ordinarily associated with increases in cAMP, yet our results show that they are required components in the antirepellent signaling pathway. Gαq
and Gβγ activity, through the activation of PLC, can produce diacylglycerol and inositol trisphosphate and thereby increase intracellular calcium 
. Thus, our present finding that both Gαq/11
and PLC are required for SDF1 antirepellent activity provides a connection between the G proteins activated by SDF1 and the calmodulin and calcium/calmodulin-stimulated cyclase that has been shown to increase cAMP downstream of SDF1. Our results are consistent with a signaling pathway () in which multiple G protein components stimulate PLC activity that induces an increase in intracellular calcium levels and leads to the activation of calmodulin. Calmodulin, in turn, activates calcium/calmodulin-stimulated adenylate cyclases, such as ADCY8, and thereby increases cAMP.
Some of the important questions that remain include how elevated cAMP levels decrease growth cone responses to repellents and the degree to which this modulation of repellent effectiveness is important in axonal pathfinding in vivo
. Both SDF1/CXCR4 activity and activity of the calmodulin-activated adenylate cyclases have a strong influence on axonal responses to the repellent slit in vivo 
. Our findings in this study suggest that activation of a wide range of GPCRs that signal through Gαi
, or Gαs
could potentially participate in axon guidance decisions.