Trafficking of proteinase-activated receptor-2 and β-arrestin-1 tagged with green fluorescent protein -: β-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 tethered ligand, once exposed, is always available to interact with its binding site. Thus, efficient mechanisms must prevent continuous activation, including receptor phosphorylation and uncoupling from G-proteins, receptor endocytosis, and lysosomal degradation. beta-Arrestins mediate uncoupling and endocytosis of certain neurotransmitter receptors, which are activated in a reversible manner. However, the role of beta-arrestins in trafficking of PARs, which are irreversibly activated, and the effects of proteases on the subcellular distribution of beta-arrestins have not been examined. We studied trafficking of PARS and beta-arrestin1 coupled to green fluorescent protein. Trypsin induced the following: (a) redistribution of beta-arrestin1 from the cytosol to the plasma membrane, where it co-localized with PAR2; (b) internalization of beta-arrestin1 and PARS into the same early endosomes; (c) redistribution of beta-arrestin1 to the cytosol concurrent with PARS translocation to lysosomes; and (d) mobilization of PARS from the Gels apparatus to the plasma membrane. Overexpression of a C-terminal fragment of beta-arrestin-(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 beta-arrestins in uncoupling of PARs.