%0 Journal Article %1 Vilardaga2001 %A Vilardaga, J. P. %A Frank, M. %A Krasel, C. %A Dees, C. %A Nissenson, R. A. %A Lohse, M. J. %D 2001 %J J Biol Chem %K 1 50 AMP/metabolism Activation Animals Arrestin/*metabolism Arrestins/metabolism Binding Binding, COS Cell Chlorides/pharmacology Competitive Compounds/pharmacology Concentration Conformation Cyclic Dose-Response Drug Enzyme Factors GTP-Binding Histidine/chemistry Hormone, Hormone/*metabolism Immunoblotting Inhibitory Inositol Ions/metabolism Kinases/*metabolism Kinetics Ligands Membrane/metabolism Mutation Parathyroid Phosphates/metabolism Phosphorylation Protein Protein-Serine-Threonine Relationship, Signal Sites Time Transduction Type Zinc Zinc/metabolism Receptor Proteins/metabolism %N 36 %P 33435-43 %T 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 %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11387315 %V 276 %X 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.

@article{Vilardaga2001, abstract = {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.}, added-at = {2010-12-14T18:12:02.000+0100}, author = {Vilardaga, J. P. and Frank, M. and Krasel, C. and Dees, C. and Nissenson, R. A. and Lohse, M. J.}, biburl = {https://www.bibsonomy.org/bibtex/207b3ee1af2677296b5bcf9db0d5393db/pharmawuerz}, endnotereftype = {Journal Article}, interhash = {ca0562018b427f35267c73b7cdd71fcb}, intrahash = {07b3ee1af2677296b5bcf9db0d5393db}, issn = {0021-9258 (Print) 0021-9258 (Linking)}, journal = {J Biol Chem}, keywords = {1 50 AMP/metabolism Activation Animals Arrestin/*metabolism Arrestins/metabolism Binding Binding, COS Cell Chlorides/pharmacology Competitive Compounds/pharmacology Concentration Conformation Cyclic Dose-Response Drug Enzyme Factors GTP-Binding Histidine/chemistry Hormone, Hormone/*metabolism Immunoblotting Inhibitory Inositol Ions/metabolism Kinases/*metabolism Kinetics Ligands Membrane/metabolism Mutation Parathyroid Phosphates/metabolism Phosphorylation Protein Protein-Serine-Threonine Relationship, Signal Sites Time Transduction Type Zinc Zinc/metabolism Receptor Proteins/metabolism}, month = {Sep 7}, note = {Vilardaga, J P Frank, M Krasel, C Dees, C Nissenson, R A Lohse, M J Research Support, Non-U.S. Gov't United States The Journal of biological chemistry J Biol Chem. 2001 Sep 7;276(36):33435-43. Epub 2001 May 31.}, number = 36, pages = {33435-43}, shorttitle = {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}, timestamp = {2010-12-14T18:21:43.000+0100}, title = {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}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11387315}, volume = 276, year = 2001 }