Differential conformational requirements for activation of G proteins and the regulatory proteins arrestin and G protein-coupled receptor kinase in the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein

After stimulation with agonist, G protein-coupled receptors (GPCRs) activat e G proteins and become phosphorylated by G protein-coupled receptor kinase s (GRKs), and most of them translocate cytosolic arrestin proteins to the c ytoplasmic membrane. Agonist-activated GPCRs are specifically phosphorylate d 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 p arathyroid hormone (PTH) receptor (PTHR), a zinc metal ion-binding site is engineered that prevents PTH-stimulated G(5) activation (Sheikh, S. P., Vil ardaga, J.-P., Baranski, T. J., Lichtarge, O., liri, T., Meng, E. C., Nisse nson, R. A., and Bourne, H. R. (1999) J. Biol. Chem. 274,17033-17041). Thes e data suggest that relative movements between H3 and H6 are critical for G . activation. Does this molecular event play a similar role in activation o f GRKs and arrestin and in PTHR-mediated G(q) activation? To answer this qu estion, 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 4 02 in H6, respectively. Both mutant receptors showed inhibition of PTH-stim ulated inositol phosphate and cAMP generation in the presence of increasing concentrations of Zn(Pi). However, the mutants showed no Zn(Pi)-dependent impairment of phosphorylation by GRK-2. Likewise, the mutants were indistin guishable from wild-type PTHR in the ability to translocate beta-arrestins/ green fluorescent protein to the cell membrane and were also not affected b y sensitivity to Zn(Il). These results suggest that agonist-mediated phosph orylation and internalization of PTHR require conformational switches of th e receptor distinct from the cAMP and inositol phosphate signaling state. F urthermore, PTHR sequestration does not appear to require G protein activat ion.