SUMMARY While the small GTPase Rac1 and its effectors are well-established mediators of mitogenic and motile signaling by tyrosine-kinase receptors and have been implicated in breast tumorigenesis, little is known regarding the exchange factors (Rac-GEFs) that mediate ErbB receptor responses. Here we identify the PIP3-Gβγ-dependent Rac-GEF P-Rex1 as an essential mediator of Rac1 activation, motility, cell growth, and tumorigenesis driven by ErbB receptors in breast cancer cells. Notably, activation of P-Rex1 in breast cancer cells requires the convergence of inputs from ErbB receptors and a Gβγ- and PI3Kγ-dependent pathway. Moreover, we identified the GPCR CXCR4 as a crucial mediator of P-Rex1/Rac1 activation in response to ErbB ligands. P-Rex1 is highly overexpressed in human breast cancers and their derived cell lines, particularly those with high ErbB2 and ER expression. In addition to the prognostic and therapeutic implications, our findings reveal an ErbB effector pathway that is crucial for breast cancer progression.

Adenosine 5-diphosphate (ADP) plays a central role in regulating platelet function by the activation of the G protein-coupled receptors P2Y 1 and P2Y 12 . Although it is well established that aggregation responses of platelets to ADP desensitize, the underlying mechanisms involved remain unclear. In this study we demonstrate that P2Y 1 -and P2Y 12 -mediated platelet responses desensitize rapidly. Furthermore, we have established that these receptors desensitize by different kinase-dependent mechanisms. G protein-coupled receptor kinase (GRK) 2 and GRK6 are both endogenously expressed in platelets. Transient overexpression of dominant-negative mutants of these kinases or reductions in endogenous GRK expression by the use of specific siRNAs in 1321N1 cells showed that P2Y 12 , but not P2Y 1 , desensitization is mediated by GRKs. In contrast, desensitization of P2Y 1 , but not P2Y 12 , is largely dependent on protein kinase C activity. This study is the first to show that both P2Y 1 and P2Y 12 desensitize in human platelets, and it reveals ways in which their sensitivity to ADP may be differentially and independently altered. IntroductionPlatelets are activated by a variety of extracellular stimuli and are an essential component of the normal response to vascular injury. Central among these stimuli is adenosine 5Ј-diphosphate (ADP), which induces multiple platelet responses and potentiates platelet aggregation to other agonists (for reviews, see Gachet 1 and Kunapuli et al 2 ). Indeed, since it was recognized 40 years ago, ADP has been regarded as a central mediator of hemostasis and thrombosis by providing a positive feedback mechanism for the activation of platelets by multiple agonists. For example, both thrombin and collagen promote ADP release from platelet-dense granules; ADP subsequently acts on purinergic receptors to reinforce platelet aggregation responses 1,2 and thrombus formation.ADP acts on 2 G protein-coupled receptors (GPCRs), P2Y 1 and P2Y 12 . 1,2 The P2Y 1 purinergic receptor was the first of the ADP receptors to be cloned. [3][4][5] This receptor is widely expressed throughout the body and couples to G q , leading to the activation of phospholipase C␤, a subsequent increase in cytosolic calcium, and the activation of protein kinase C (PKC). The P2Y 12 receptor was only recently identified 6 and is the target of the clinically effective antithrombotic drugs clopidogrel and ticlopidine. P2Y 12 couples through G i to the inhibition of adenylyl cyclase and the activation of PI3-kinase. On the basis of pharmacologic and genetic studies, it is now accepted that P2Y 1 is required for platelet activation by ADP, whereas P2Y 12 is important in synergizing with P2Y 1 or other G q -coupled receptors to induce platelet activation by ADP and other agonists playing a major role in stabilizing platelet thrombi in vivo.Given the established crucial role of ADP in platelet activation, it is likely that the responsiveness of platelets to ADP is tightly regulated, and knowledge of the mechanisms responsible for t...