Abstract
Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) activated by thrombin, directly couples with Gαq/11, Gαi2, Gα12/13 to transduce signals. After activation by irreversible cleavage at the specific site of its N-terminus, PAR1 was internalized via clathrin-dependent endocytosis and sorted to lysosomes for degradation. Caveolin-1, a major structure protein enriched in caveolae, plays important roles in regulation of signal transduction and internalization of GPCRs. However, it is not clear whether PAR1, its downstream G protein alpha subunits, and extracellular signal-regulated kinase (ERK) reside in caveolae before and after the receptor activation. In this study, the caveolin-enriched fractions prepared by the detergent-free sucrose gradient centrifugation were used to examine the distribution of these proteins. Before PAR1 activation, the receptor was highly confined in the caveolin-enriched fractions. Gαq and Gα13 were predominantly found in the caveolin-enriched fractions. A large portion of Gαi, Gα12, and ERK was observed in the high-density fractions. After PAR1 activation, the receptor was moved out of the caveolin-enriched fractions to be degraded. Gαq and Gα13 were still immobilized in the caveolin-enriched fractions. Gαi was moved in and then moved out of the caveolin-enriched fractions, whereas Gα12 was gradually moved out of the caveolin-enriched fractions. Furthermore, phosphorylated ERK was found in the fraction 4 to 6 and 7 to 12, while ERK was gradually accumulated in the caveolin-enriched fractions. These results indicate that lipid raft/caveolae may act as major plasma membrane microdomains where PAR1 is localized to modulate its downstream signal transduction.