Abstract
Sequestration of porcine muscarinic acetylcholine receptor m2 subtypes (m2 receptors) expressed in COS-7 cells is facilitated by coexpression of G protein-coupled receptor kinases 2 (GRK2). We examined the effect of coexpression of GRK2, GRK4δ, GRK5 and GRK6 on sequestration of human m1—m5 receptors expressed in COS-7 cells, which was assessed as loss of [3H]N-methylscopolamine binding activity from the cell surface. Sequestration of m4 receptors as well as m2 receptors was facilitated by coexpression of GRK2 and attenuated by coexpression of the dominant negative form of GRK2 (DN-GRK2). Sequestration of m3 and m5 receptors also was facilitated by coexpression of GRK2 but not affected by coexpression of DN-GRK2. On the other hand, proportions of sequestered m1 receptors were not significantly different with coexpression of GRK2 and DN-GRK2. GRK4δ, GRK5 and GRK6 did not facilitate sequestration of m1—m5 receptors in COS-7 cells, except that the sequestration of m2 receptors tended to be facilitated by coexpression of GRK4δ, GRK5 and GRK6. However, coexpression of GRK4δ, GRK5, but not GRK6, in BHK-21 cells facilitated sequestration of m2, but not m3, receptors. These results indicate that the effect of GRK2 to facilitate receptor sequestration is not restricted to m2 receptors but is generalized to other muscarinic receptors except m1 receptors and that other kinases, including GRK4δ, GRK5 and endogenous kinase(s) in COS-7 cells, also contribute to sequestration of m2 and m4 receptors.
Footnotes
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Send reprint requests to: Dr. Hirofumi Tsuga, National Institute of Industrial Health, 6-21-1, Nagao, Tama-ku, Kawasaki, Kanagawa, 214, Japan. E-mail: tsuga{at}m.u-tokyo.ac.jp
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↵1 This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan, Japan Society for the Promotion of Science and Japan Health Science Foundtion.
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↵2 Present address: National Institute of Industrial Health, 6-21-1, Nagao, Tamaku, Kawasaki, Kanagawa, 214, Japan.
- Abbreviations:
- PBS
- phosphate-buffered saline
- GRK
- G protein-coupled receptor kinase
- DN-GRK2
- dominant negative form of GRK2
- G protein
- guanine nucleotide-binding regulatory protein
- NMS
- N-methylscopolamine
- SDS
- sodium dodecyl sulfate
- PAGE
- polyacrylamide gel electrophoresis
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- GST
- glutathione-S-transferase
- Received July 9, 1997.
- Accepted November 13, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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