Role of signal transduction in internalization of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein

For G protein-coupled receptors, limited information is available on the ro le of agonist binding or of the second-messenger products of receptor signa ling on receptor endocytosis. We explored this problem using the opossum PT H/PTH-related protein (PTHrP) receptor, a prototypical Class II G protein-c oupled receptor, as a model. In one approach, we evaluated the endocytic pr operties of mutated forms of the opossum PTH/PTHrP receptor that we had pre viously shown to be impaired in their ability to initiate agonist-induced s ignaling when expressed in COS-7 cells. A point mutation in the third cytop lasmic loop (K382A) that severely impairs PTH/PTHrP receptor signaling sign ificantly reduced internalization, whereas two mutant receptors that displa yed only partial defects in signaling were internalized normally. To explor e more directly the role of second-messenger pathways, we used a cleavable biotinylation method to assess endocytosis of the wild-type receptor stably expressed in human embryonic kidney (HEK) 293 cells. A low rate of constit utive internalization was detected (<5% over a 30-min incubation at 37 C); the rate of receptor internalization was enhanced about 10-fold by the rece ptor agonists PTH(1-34) or PTHrP(1-34), whereas the receptor antagonist PTH (7-34) had no effect. Forskolin treatment produced a minimal increase in co nstitutive receptor endocytosis, and the protein kinase (PK)-A inhibitor H- 89 failed to block agonist-stimulated endocytosis. Similarly, activation of PK-C, by treatment with phorbol 12-myristate 13-acetate, elicited only a m inimal increase in constitutive receptor endocytosis; and blockade of the P K-C pathway, by treatment with a bisindolylmaleimide, failed to inhibit ago nist-induced receptor endocytosis. Immunofluorescence confocal microscopic studies of PTH/PTHrP receptor internalization confirmed the results using r eceptor biotinylation. These findings suggest that: 1) agonist binding is r equired for the efficient endocytosis of the PTH/PTHrP receptor; 2) recepto r activation (agonist-induced receptor conformational change) and/or coupli ng to G proteins plays a critical role in receptor internalization; and 3) activation of PK-A and PK-C is neither necessary nor sufficient for agonist -stimulated receptor internalization.