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

• Published in: The Journal of biological chemistry 2001-09, Vol.276 (36), p.33435-33443
• Main Authors: Vilardaga, Jean-Pierre, Frank, Monika, Krasel, Cornelius, Dees, Christian, Nissenson, Robert A., Lohse, Martin J.
• Format: Article
• Language: English
• Subjects: Animals; arrestin; Arrestin - metabolism; Arrestins - metabolism; beta-Arrestins; Binding Sites; Binding, Competitive; Cell Membrane - metabolism; Chlorides - pharmacology; COS Cells; Cyclic AMP - metabolism; Dose-Response Relationship, Drug; Enzyme Activation; GTP-Binding Proteins - metabolism; Histidine - chemistry; Immunoblotting; Inhibitory Concentration 50; Inositol Phosphates - metabolism; Ions - metabolism; Kinetics; Ligands; Mutation; Phosphorylation; Protein Binding; Protein Conformation; Protein-Serine-Threonine Kinases - metabolism; Receptor, Parathyroid Hormone, Type 1; Receptors, Parathyroid Hormone - metabolism; Signal Transduction; Time Factors; Zinc - metabolism; Zinc Compounds - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M011495200

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 Gs 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 Gs activation. Does this molecular event play a similar role in activation of GRK and arrestin and in PTHR-mediated Gq 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 β-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.