After stimulation with agonist, G protein-coupled receptors (GPCRs)
activate G proteins and become phosphorylated by G protein-coupled
receptor kinases (GRKs), and most of them translocate cytosolic arrestin
proteins to the cytoplasmic membrane. Agonist-activated GPCRs are
specifically phosphorylated by GRKs and are targeted for endocytosis
by arrestin proteins, suggesting a connection between GPCR conformational
changes and interaction with GRKs and arrestins. Previously, we showed
that by substitution of histidine for residues at the cytoplasmic
side of helix 3 (H3) and helix 6 (H6) of the parathyroid hormone
(PTH) receptor (PTHR), a zinc metal ion-binding site is engineered
that prevents PTH-stimulated G(s) activation (Sheikh, S. P., Vilardaga,
J.-P., Baranski, T. J., Lichtarge, O., Iiri, T., Meng, E. C., Nissenson,
R. A., and Bourne, H. R. (1999) J. Biol. Chem. 274, 17033-17041).
These data suggest that relative movements between H3 and H6 are
critical for G(s) activation. Does this molecular event play a similar
role in activation of GRK and arrestin and in PTHR-mediated G(q)
activation? To answer this question, we utilized the two previously
described mutant forms of PTHR, H401 and H402, which contain a naturally
present histidine residue at position 301 in H3 and a second substituted
histidine residue at positions 401 and 402 in H6, respectively. Both
mutant receptors showed inhibition of PTH-stimulated inositol phosphate
and cAMP generation in the presence of increasing concentrations
of Zn(II). However, the mutants showed no Zn(II)-dependent impairment
of phosphorylation by GRK-2. Likewise, the mutants were indistinguishable
from wild-type PTHR in the ability to translocate beta-arrestins/green
fluorescent protein to the cell membrane and were also not affected
by sensitivity to Zn(II). These results suggest that agonist-mediated
phosphorylation and internalization of PTHR require conformational
switches of the receptor distinct from the cAMP and inositol phosphate
signaling state. Furthermore, PTHR sequestration does not appear
to require G protein activation.