Abstract
Abstract Protease-activated receptor 1 (PAR1), a G protein-coupled receptor, is activated by thrombin cleavage at the amino-terminal domain of the receptor. Upon activation, PAR1 couples to several G proteins to transduce signals from cell membrane to cells. Since the activation is irreversible, PAR1 has to be rapidly terminated its signaling by uncoupling from G proteins, internalized to endosome and degraded in lysosomes. PAR1-induced cell proliferation has been reported to be mediated by Src kinase activation. In addition, overexpression of PAR1 can induce cell transformation and cell invasion in several cells. In breast carcinoma cells, PAR1 is overexpressed and fails to be degraded, which contributes prolonged signaling and cell invasion. Thus, the regulation of PAR1 trafficking, activation, and downregulation has critical roles in PAR1-induced cell proliferation and cell invasion. Caveolae, specialized plasmalemmal microdomains, belong to one subset of lipid rafts but can be distinguished by the presence of the cholesterol binding protein caveolin-1. Several G protein-coupled receptors are internalized via caveolae and transduce their signals in caveolae. In addition, caveolae play a role in cell migration. However, the involvement of caveolae in PAR1 trafficking, PAR1-mediated signaling and PAR1-induced cell migration remained unclear. In this study, the involvement of caveolae in both the localization and internalization of PAR1 were first examined. Analysis of the cells by discontinuous-sucrose gradient centrifugation shows that PAR1 was partially localized in the caveolin-1-enriched fraction. However, cholesterol extraction by methyl-□-cyclodextrin to disrupt caveolae could not inhibit PAR1 internalization, indicating that caveolae are not involved in PAR1 internalization. Since some receptors can induce caveolin-1 phosphorylation on tyrosine 14, the involvement of caveolae in PAR1-induced phosphorylation of caveolin-1 was then examined. It was found that caveolin-1 was phosphorylated at tyrosine 14 by a Gi-linked Src kinase and p38 mitogen-activated protein kinase pathways. Phosphocaveolin-1 but not caveolin-1 with mutation at tyrosine 14 could bind to c-terminal Src kinase (Csk). The recruitment of Csk by phosphocaveolin-1 further resulted in a rapid decrease in Src kinase activity, indicating that phosphocaveolin-1 represents a novel effector of PAR1 to downregulate Src kinase activity. Moreover, the involvement of caveolae in PAR1-induced migration has been investigated by wound healing assay in MDA-MB-231 cells. Cholesterol extraction by methyl-□-cyclodextrin to disrupt caveolae inhibited PAR1-induced cell migration and cholesterol repletion restored PAR1-induced cell migration, indicating that caveolae are involved in PAR1-induced cell migration. The localization of Gi proteins, ERK1/2 and PI3K, downstream signaling molecules of PAR1, in caveolae and their involvement in PAR1-induced cell migration were also examined by discontinuous-sucrose gradient centrifugation and wound healing assay. A small portion of Gi proteins were localized in caveolin-1-enriched fraction before and after PAR1 activation. Of interest, activation of PAR1 resulted in the redistribution of a portion of phosphorylated ERK1/2 to caveolae. However, PI3K did not appear at caveolin-1-enriched fraction before and after PAR1 activation. Inactivation of signaling molecules localized in caveolae, Gi proteins or ERK1/2, but not PI3K inhibited PAR1-induced cellular migration. PAR1-induced ERK1/2 activation was also partially inhibited by disruption of caveolae, indicating PAR1-induced ERK1/2 activation was partially mediated by caveolae. These results suggest that caveolae and PAR1’s downstream signaling molecules, Gi and ERK1/2, localized in caveolae are involved in PAR1-induced cell migration. Taken together, PAR1-induced Src activity is regulated by phosphocaveolin-1, implicating that caveolae can regulate PAR1-induced cell proliferation. In addition, caveolae and signaling molecules downstream of PAR1 concentrated in caveolae are involved in PAR1-induced cell migration, indicating that caveolae regulate PAR1-induced cell migration. These results show that caveolae are involved in regulation of PAR1-mediated signaling and PAR1-induced cell migration. The findings also provide new insight of how caveolae play a role in PAR1-induced tumor formation.