Regulated Shedding of PAR1 N-terminal Exodomain from Endothelial Cells

Abstract

G protein-coupled receptors can trigger metalloproteinase-dependent shedding of proteins from the cell surface. We now report that G protein-coupled receptors can themselves undergo regulated metalloproteinasedependent shedding. The N-terminal exodomain of protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, displayed regulated shedding in endothelial cells, which normally express this receptor. Cleavage occurred at a site predicted to render the receptor unresponsive to thrombin. A chimeric protein in which the N-terminal exodomain of PAR1 was fused to an unrelated transmembrane segment was shed as efficiently as PAR1, shedding of both proteins was stimulated by phorbol ester and by a PAR1 agonist. TNF␣ protease inhibitor-2 (TAPI-2), phenanthroline, and tissue inhibitor of metalloproteinase-3 (TIMP-3) but not TIMP-1 or -2 inhibited such shedding. These and other data suggest that the information that specifies PAR1 shedding resides within its N-terminal exodomain rather than its heptahelical segment, that activation of protein kinase C or of PAR1 itself can stimulate PAR1 shedding in trans, and that ADAM17/TACE or a metalloproteinase with similar properties mediates PAR1 shedding. Regulated shedding reduced the amount of cell surface PAR1 available for productive cleavage by thrombin by half or more, but thus far we have been unable to demonstrate an effect of PAR1 shedding on cellular responsiveness to thrombin. Nonetheless, regulated shedding of G protein-coupled receptors represents a new mechanism by which signaling by this important class of receptors might be modulated.

Human β1-Adrenergic Receptor Is Subject to Constitutive and Regulated N-terminal Cleavage

“…Consistent with the findings obtained for the h␤ 1 AR, the endothelin B, V 2 vasopressin, and thyrotropin receptors are cleaved in an activationdependent manner following agonist binding (36 -38, 42), although conflicting reports have been published for the thyrotropin receptor (44). On the other hand, the thrombin-mediated cleavage of PAR-1 has been shown to take place following protein kinase C activation (40), and furthermore, it has been shown that matrix metalloproteinase I can mediate the cleavage and activation of PAR-1, which leads to invasion and metastasis of tumor cells (41). Otherwise, the functional consequences of metalloproteinase-mediated cleavage have not been fully revealed for any other family A GPCRs.…”

“…for endothelin B (35,36) and thyrotropin (37)(38)(39) receptors, and for the protease-activated receptor-1 (PAR-1) (40,41), which is the main GPCR for thrombin. The V 2 vasopressin receptor, on the other hand, has been shown to be cleaved at the interface between the second transmembrane domain and the first extracellular loop (42,43).…”

Protease-activated receptor signalling, endocytic sorting and dysregulation in cancer

“…Several other proteases are also capable of cleaving and activating PAR2, as well as other PARs, but whether these proteases function as physiological regulators in vivo awaits validation. In addition, some proteases, including certain MMPs, disable PARs by cleaving downstream of the activation site, resulting in loss of the tethered ligand domain (Ludeman et al, 2004), and thus revealing a potentially important mechanism for regulation of PAR signalling in various cell types.…”

The Parkinson's-disease-associated receptor GPR37 undergoes metalloproteinase-mediated N-terminal cleavage and ectodomain shedding

“…The cleavage of this GPCR leads to the formation of a two-subunit receptor structure that is held together by disulphide bonds, the reduction of which can lead to the shedding of the receptor ectodomain (Couet et al, 1996). In addition to TSHR, the angiotensin II type 1 receptor and the protease-activated receptor-1 are the only other class A GPCRs, for which ectodomain shedding has been demonstrated (Ludeman et al, 2004;Cook et al, 2007).…”