Integrins regulate adhesion-dependent growth, survival and invasion of tumor cells. In particular, expression of integrin αvβ3 is associated with progression of a variety of human tumors. Here, we reveal a novel adhesion-independent role for integrin αvβ3 in pancreatic cancer and other carcinomas. Specifically, αvβ3 expressed in carcinoma cells enhanced anchorage-independent tumor growth in vitro and increased lymph node metastases in vivo. This required recruitment of c-src to the β3 integrin cytoplasmic tail, leading to c-src activation, crk-associated substrate (CAS) phosphorylation and tumor cell survival that, surprisingly, was independent of cell adhesion or focal adhesion kinase (FAK) activation. Reduced expression of endogenous αvβ3 or c-src not only suppressed anchorage-independent growth, but also decreased metastasis in vivo, yet did not affect migration/invasion. These data define an unexpected role for an integrin as a mediator of anchorage-independence suggesting that an αvβ3/c-src signaling module may account for the aggressive behavior of αvβ3-expressing tumors in man.

Heterotrimeric G proteins mediate the earliest step in cell responses to external events by linking cell surface receptors to intracellular signaling pathways. Gz is a member of the Gi family of G proteins that is prominently expressed in platelets and brain. Here, we show that deletion of the ␣ subunit of Gz in mice: (i) impairs platelet aggregation by preventing the inhibition of cAMP formation normally seen at physiologic concentrations of epinephrine, and (ii) causes the mice to be more resistant to fatal thromboembolism. Loss of Gz␣ also results in greatly exaggerated responses to cocaine, reduces the analgesic effects of morphine, and abolishes the effects of widely used antidepressant drugs that act as catecholamine reuptake inhibitors. These changes occur despite the presence of other Gi␣ family members in the same cells and are not accompanied by detectable compensatory changes in the level of expression of other G protein subunits. Therefore, these results provide insights into receptor selectivity among G proteins and a model for understanding platelet function and the effects of psychoactive drugs.H eterotrimeric guanine nucleotide binding proteins (G proteins) comprised of ␣, ␤, and ␥ subunits mediate diverse cellular responses by linking receptors on the cell surface to intracellular signaling pathways. At least 20 human genes are known to encode GTP-binding ␣ subunits. Half of these are members of the G i␣ family, including the ubiquitously expressed and nearly identical G i1␣ , G i2␣ , and G i3␣ , as well as several with restricted expression, such as G z␣ , G o␣ , and transducin. The best described effector for most G i family members is adenylyl cyclase. However, others exist as well, including cGMP phosphodiesterase, ion channels, phospholipase C, and Rap1GAP (1-5). One unresolved issue is the need for such a multiplicity of G i␣ family members, many of which are commonly expressed within the same cell. One approach to addressing this question has been the development of mice that lack individual family members. Of the three broadly expressed members of the family, mice lacking G i2␣ have the most striking phenotype with abnormalities of T cell function and thymocyte maturation, as well as an increased susceptibility to develop inflammatory bowel disease (6-8). Deletion of G i1␣ or G i3␣ , on the other hand, has not been reported to produce an obvious effect. Clearly, however, if there are functional differences among these and the other G i family members, then receptor selection among them is potentially a major determinant for cellular responses.Of the 10 known members of the G i␣ family, the sequence of G z␣ bears the least similarity to the others. G z␣ has a limited distribution in humans with prominent expression in blood platelets and selected areas of the brain. Like other members of the family, G z␣ has been shown to inhibit cAMP formation by adenylyl cyclase when over-expressed (9), but it is not known whether this is part of its role in vivo. Similarly, in over-expression systems, ...

Eph kinases are receptor tyrosine kinases whose ligands, the ephrins, are also expressed on the surface of cells. Interactions between Eph kinases and ephrins on adjacent cells play a central role in neuronal patterning and vasculogenesis. Here we examine the expression of ephrins and Eph kinases on human blood platelets and explore their role in the formation of the hemostatic plug. The results show that human platelets express EphA4 and EphB1, and the ligand, ephrinB1. Forced clustering of EphA4 or ephrinB1 led to cytoskeletal reorganization, adhesion to fibrinogen, and ␣-granule secretion. Clustering of ephrinB1 also caused activation of the Ras family member, Rap1B. In platelets that had been activated by ADP and allowed to aggregate, EphA4 formed complexes with two tyrosine kinases, Fyn and Lyn, and the cell adhesion molecule, L1. Blockade of Eph͞ephrin interactions prevented the formation of these complexes and caused platelet aggregation at low ADP concentrations to become more readily reversible. We propose that when sustained contacts between platelets have occurred in response to agonists such as collagen, ADP, and thrombin, the binding of ephrins to Eph kinases on adjacent platelets provides a mechanism to perpetuate signaling and promote stable platelet aggregation.

The recent identification of two new thrombin receptors, PAR3 and PAR4, led us to re-examine the basis for endothelial cell responses to thrombin. Human umbilical vein endothelial cells (HUVEC) are known to express PAR1 and the trypsin/tryptase receptor, PAR2. Northern blots detected both of those receptors and, to a lesser extent, PAR3, but PAR4 message was undetectable and there was no response to PAR4 agonist peptides. To determine whether PAR3 or any other receptor contributes to thrombin signaling in HUVEC, PAR1 cleavage was blocked with two selective antibodies and PAR1 activation was inhibited with the antagonist, BMS200261. The antibodies completely inhibited HU-VEC responses to thrombin, but BMS200261 was only partly effective, even though separate studies established that the antagonist completely inhibits PAR1 signaling at the concentrations used. Since peptides mimicking the PAR1 tethered ligand domain can also activate PAR2, we asked whether the remaining thrombin response in the presence of the antagonist could be due in part to the intermolecular transactivation of PAR2 by cleaved PAR1. Evidence that transactivation can occur was obtained in COS-7 cells co-expressing PAR2 and a variant of PAR1 that can be cleaved, but not signal. There was a substantial response to thrombin only in cells expressing both receptors. Conversely, in HUVEC, complete blockade of the thrombin response by the PAR1 antagonist occurred only when signaling through PAR2 was also blocked. From these observations we conclude that 1) PAR1 is the predominant thrombin receptor expressed in HUVEC and cleavage of PAR1 is required for endothelial cell responses to thrombin; 2) although PAR3 may be expressed, there is still no evidence that it mediates thrombin responses; 3) PAR4 is not expressed on HUVEC; and 4) transactivation of PAR2 by cleaved PAR1 can contribute to endothelial cell responses to thrombin, particularly when signaling through PAR1 is blocked. Such transactivation may limit the effectiveness of PAR1 antagonists, which compete with the tethered ligand domain rather than preventing PAR1 cleavage.

The ability of activated platelets to adhere to each other at sites of vascular injury depends on the integrin ␣IIb␤3. However, as aggregation continues, other signaling and adhesion molecules can contribute as well. We have previously shown that human platelets express on their surface the Eph receptor kinases EphA4 and EphB1 and the Eph kinase ligand ephrinB1. We now show that EphA4 is physically associated with ␣IIb␤3 in resting platelets, increases its surface expression when platelets are activated, and colocalizes with ␣IIb␤3 at sites of contact between platelets. We also show that Eph͞ephrin interactions can support the stable accumulation of platelets on collagen under flow and contribute to postengagement ''outside-in'' signaling through ␣IIb␤3 by stabilizing platelet aggregates and facilitating tyrosine phosphorylation of the ␤3 cytoplasmic domain. ␤3 phosphorylation allows myosin to bind to ␣IIb␤3 and clot retraction to occur. The data support a model in which the onset of aggregation permits Eph͞ephrin interactions to occur, after which signaling downstream from ephrinB1 and its receptors favors continued growth and stability of the thrombus by several mechanisms, including positive effects on outside-in signaling through ␣IIb␤3.