Abstract
Human muscarinic acetylcholine receptor m1 subtypes (m1 receptors) were expressed in and purified from insect Sf9 cells and then subjected to phosphorylation by G protein-coupled receptor kinase 2 (GRK2) expressed in and purified from Sf9 cells and by protein kinase C purified from rat brain (a mixture of alpha, beta, and gamma types, PKC). The m1 receptor was phosphorylated by either GRK2 or PKC in an agonist-dependent or independent manner, respectively. G protein beta gamma subunits stimulated the phosphorylation by GRK2 but did not affect the phosphorylation by PKC. The number of incorporated phosphates was 4.6 and 2.8 mol/mol of receptor for phoshorylation by GRK2 and PKC, respectively. The number of incorporated phosphates was 7.5 mol/mol receptor for phosphorylation by GRK2 followed by PKC, but was 5.8 mol/mol of receptor for the phosphorylation by PKC followed by GRK2. Major sites phosphorylated by GRK2 and PKC were located in the third intracellular loop and the carboxyl-terminal tail, respectively. These results indicate that GRK2 and PKC phosphorylate different sites of m1 receptors and that the phosphorylation by PKC partially inhibits the phosphorylation by GRK2, probably by affecting activation of GRK2 by agonist-bound receptors.
Highlights
Muscarinic acetylcholine receptors consist of five subtypes
In the present paper we have shown that human m1 receptors are phosphorylated in an agonist-dependent manner by G protein-coupled receptor kinase 2 (GRK2) and independent manner by protein kinase C (PKC)
The number of phosphorylation sites were estimated to be 4 –5 for phosphorylation by GRK2 and 2–3 for phosphorylation by PKC. These phosphorylation sites appear to be different from each other, because the sum of sites phosphorylated by GRK2 and PKC was not significantly different from the number of sites phosphorylated following sequential phosphorylation by GRK2 and PKC. This conclusion was further supported by the finding that major phosphorylation bands obtained by trypsin treatment of m1 receptors phosphorylated by GRK2 are different from those obtained by the same treatment of m1 receptors phosphorylated by PKC
Summary
Materials—Human m1 muscarinic receptors were expressed in and purified from Sf9 cells by single step affinity chromatography as described previously [35, 36]. The void volume fraction was mixed with 1.1 ml of HEN solution containing 5 mM dithiothreitol and 1 mM carbamylcholine and with 0.5 ml of 50% (w/v) polyethylene glycol 6000 (PEG), kept for 10 min at room temperature, and centrifuged for 30 min at 50,000 rpm. A standard assay tube for phosphorylation of m1 receptors by GRK2 contained the reconstituted vesicle (1 l; 1.8 – 4.0 nM m1 receptors and 9 – 40 nM Go in final concentrations), 1 mM carbamylcholine or 10 M atropine, 100 M GTP, 1 or 10 M [␥-32P]ATP (2 ϫ 105 cpm/tube, 1–10 cpm/fmol), and purified GRK2 (13 nM) in a medium of 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM EDTA, 0.5 mM EGTA (total volume, 40 l). Molecular weights of small peptides were estimated from the mobility of marker proteins and a phosphorylated synthetic peptide corresponding to the carboxyl terminus of m1 receptors (residue 435– 460)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
