Trafficking of proteinase-activated receptor-2 and beta-arrestin-1 tagged with green fluorescent protein - beta-arrestin-dependent endocytosis of a proteinase receptor

Proteases cleave proteinase-activated receptors (PARs) to expose N-terminal tethered ligands that bind and activate the cleaved receptors. The tethere d ligand, once exposed, is always available to interact with its binding si te. Thus, efficient mechanisms must prevent continuous activation, includin g receptor phosphorylation and uncoupling from G-proteins, receptor endocyt osis, and lysosomal degradation. beta-Arrestins mediate uncoupling and endo cytosis of certain neurotransmitter receptors, which are activated in a rev ersible manner. However, the role of beta-arrestins in trafficking of PARs, which are irreversibly activated, and the effects of proteases on the subc ellular distribution of beta-arrestins have not been examined. We studied t rafficking of PARS and beta-arrestin1 coupled to green fluorescent protein. Trypsin induced the following: (a) redistribution of beta-arrestin1 from t he cytosol to the plasma membrane, where it co-localized with PAR2; (b) int ernalization of beta-arrestin1 and PARS into the same early endosomes; (c) redistribution of beta-arrestin1 to the cytosol concurrent with PARS transl ocation to lysosomes; and (d) mobilization of PARS from the Gels apparatus to the plasma membrane. Overexpression of a C-terminal fragment of beta-arr estin-(319-418), which interacts constitutively with clathrin but does not bind receptors, inhibited agonist-induced endocytosis of PARS. Our results show that p-arrestins mediate endocytosis of PARS and support a role for be ta-arrestins in uncoupling of PARs.