Zm. Huang et al., Role of signal transduction in internalization of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein, ENDOCRINOL, 140(3), 1999, pp. 1294-1300
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.