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Gq-coupled purinergic receptors inhibit insulin-like growth factor-I/phosphoinositide 3-kinase pathway-dependent keratinocyte migration.

ABSTRACT: Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Galpha((q/11))-coupled-P2Y(2) purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y(2) receptors by extracellular UTP inhibits the IGF-I-induced p110alpha-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Galpha((q/11))-and not G((i/o))-independently of phospholipase Cbeta. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110alpha mutant, in a Galpha(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.

SUBMITTER: Taboubi S

PROVIDER: S-EPMC2836975 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In superior cervical ganglion (SCG) neurons, stimulation of M(1) receptors (M(1)Rs) produces a distinct pattern of modulation of N-type calcium (N-) channel activity, enhancing currents elicited with negative test potentials and inhibiting currents elicited with positive test potentials. Exogenously applied arachidonic acid (AA) reproduces this profile of modulation, suggesting AA functions as a downstream messenger of M(1)Rs. In addition, techniques that diminish AA's concentration during M(1)R stimulation minimize N-current modulation. However, other studies suggest depletion of phosphatidylinositol-4,5-bisphosphate during M(1)R stimulation suffices to elicit modulation. In this study, we used an expression system to examine the physiological mechanisms regulating modulation. We found the beta subunit (Ca(V)beta) acts as a molecular switch regulating whether modulation results in enhancement or inhibition. In human embryonic kidney 293 cells, stimulation of M(1)Rs or neurokinin-1 receptors (NK-1Rs) inhibited activity of N channels formed by Ca(V)2.2 and coexpressed with Ca(V)beta1b, Ca(V)beta3, or Ca(V)beta4 but enhanced activity of N channels containing Ca(V)beta2a. Exogenously applied AA produced the same pattern of modulation. Coexpression of Ca(V)beta2a, Ca(V)beta3, and Ca(V)beta4 recapitulated the modulatory response previously seen in SCG neurons, implying heterogeneous association of Ca(V)beta with Ca(V)2.2. Further experiments with mutated, chimeric Ca(V)beta subunits and free palmitic acid revealed that palmitoylation of Ca(V)beta2a is essential for loss of inhibition. The data presented here fit a model in which Ca(V)beta2a blocks inhibition, thus unmasking enhancement. Our discovery that the presence or absence of palmitoylated Ca(V)beta2a toggles M(1)R- or NK-1R-mediated modulation of N current between enhancement and inhibition identifies a novel role for palmitoylation. Moreover, these findings predict that at synapses, modulation of N-channel activity by M(1)Rs or NK-1Rs will fluctuate between enhancement and inhibition based on the presence of palmitoylated Ca(V)beta2a.

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Gq-coupled purinergic receptors inhibit insulin-like growth factor-I/phosphoinositide 3-kinase pathway-dependent keratinocyte migration.

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