Involvement of G protein-coupled receptor kinase-6 in desensitization of CGRP receptors

Eur J Pharmacol. 2000 Sep 1;403(1-2):1-7. doi: 10.1016/s0014-2999(00)00419-2.


This investigation was undertaken to study the mechanisms of calcitonin gene-related peptide (CGRP)-mediated desensitization using recombinant porcine CGRP receptors stably expressed in human embryonic kidney (HEK-293) cells. Pretreatment of these cells with human alphaCGRP resulted in an approximately 60% decrease in CGRP-stimulated adenylyl cyclase activity and an approximately 10-fold rightward shift in the dose-response curve of CGRP. This effect was rapid (t(1/2) approximately 5 min) and was accompanied by a significant decrease in [125I]CGRP binding to membrane preparations from CGRP-pretreated cells. In contrast, CGRP pretreatment had no effect on isoproterenol- or forskolin-stimulated adenylyl cyclase activity in these cells. The potential involvement of protein kinase A or protein kinase C in CGRP-mediated desensitization was studied using selective inhibitors or activators of these kinases. Pretreatment of the cells with forskolin (adenylyl cyclase activator) or phorbol dibutyrate (protein kinase C activator) had no effect on CGRP-mediated adenylyl cyclase activity and did not influence CGRP-mediated desensitization. However, pretreatment of the cells with 2-(8-[(dimethylamino)methyl]-6,7,8, 9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methylindol-3-yl)m aleimide hydrochloride (Ro 32-0432) (a potent inhibitor of protein kinase C) resulted in significant attenuation of CGRP-mediated desensitization with an IC(50) approximately 3 microM. To establish whether this effect might be due to inhibition of other protein kinases by Ro 32-0432, its effect was tested against several G protein-coupled receptor kinases (GRKs). Ro 32-0432 was found to inhibit GRK2, GRK5, and GRK6 with IC(50) values of 29, 3.6, and 16 microM, respectively, suggesting that its effect on CGRP-mediated desensitization might be a result of GRK inhibition. To further test this hypothesis, as well as the potential GRK specificity, the cells were treated with antisense oligonucleotides to GRK2, GRK5, and GRK6. While GRK2 and GRK5 antisense nucleotides had no effect on CGRP-mediated desensitization, the GRK6 antisense nucleotide treatment significantly reversed CGRP-mediated desensitization. These results suggest the involvement of GRK6 in CGRP-mediated desensitization in HEK-293 cells.

MeSH terms

  • Adenylyl Cyclases / metabolism
  • Animals
  • Binding, Competitive / drug effects
  • Calcitonin Gene-Related Peptide / metabolism
  • Calcitonin Gene-Related Peptide / pharmacology
  • Calcitonin Gene-Related Peptide Receptor Antagonists
  • Cell Line
  • Cyclic AMP-Dependent Protein Kinases / genetics
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • DNA, Antisense / pharmacology
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / pharmacology
  • G-Protein-Coupled Receptor Kinase 5
  • G-Protein-Coupled Receptor Kinases
  • Humans
  • Indoles / pharmacology
  • Iodine Radioisotopes
  • Membranes / drug effects
  • Membranes / metabolism
  • Peptide Fragments / pharmacology
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Pyrroles / pharmacology
  • Radioligand Assay
  • Receptors, Calcitonin Gene-Related Peptide / metabolism*
  • Swine
  • beta-Adrenergic Receptor Kinases


  • Calcitonin Gene-Related Peptide Receptor Antagonists
  • DNA, Antisense
  • Enzyme Inhibitors
  • Indoles
  • Iodine Radioisotopes
  • Peptide Fragments
  • Pyrroles
  • Receptors, Calcitonin Gene-Related Peptide
  • calcitonin gene-related peptide (8-37)
  • Ro 32-0432
  • Protein-Serine-Threonine Kinases
  • Cyclic AMP-Dependent Protein Kinases
  • beta-Adrenergic Receptor Kinases
  • G-Protein-Coupled Receptor Kinase 5
  • G-Protein-Coupled Receptor Kinases
  • G-protein-coupled receptor kinase 6
  • GRK5 protein, human
  • Adenylyl Cyclases
  • Calcitonin Gene-Related Peptide