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) 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.
Vilardaga
J P
JP
Department of Pharmacology, Institute of Pharmacology and Toxicology, University of Würzburg, D-97078 Würzburg, Germany.
Frank
M
M
Krasel
C
C
Dees
C
C
Nissenson
R A
RA
Lohse
M J
MJ
D000818:Animals; D019393:Arrestin / Q000378:metabolism*; D019390:Arrestins / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D007295:Inositol Phosphates / Q000378:metabolism; D007477:Ions / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D015032:Zinc / Q000378:metabolism; D017967:Zinc Compounds / Q000494:pharmacology; D000071557:beta-Arrestins
D019393:Arrestin; D019390:Arrestins; D002712:Chlorides; D007295:Inositol Phosphates; D007477:Ions; D008024:Ligands; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D017967:Zinc Compounds; D000071557:beta-Arrestins; D006639:Histidine; C016837:zinc chloride; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases; D019204:GTP-Binding Proteins; D015032:Zinc