Differences in desensitization between human beta 1- and beta 2-adrenergic receptors stably expressed in transfected hamster cells.
It is well-established that agonist-mediated desensitization of the beta 2-adrenergic receptor (beta 2AR) involves its phosphorylation by protein kinase A (PKA) and the beta AR kinase (beta ARK). The phosphorylated receptor is less efficient at mediating agonist stimulation of adenylyl cyclase activity. The result is an increase in the concentration of agonist required for half-maximal stimulation (EC50) and a reduction in maximal stimulation (Vmax). As less is known about desentization of the human beta 1 AR, we compared the desensitization pattern of human beta 1 AR and beta 2AR stably expressed in two different hamster cell lines: Chinese hamster ovary (CHO), and Chinese hamster fibroblast (CHW). Following agonist treatment, all of the cell lines exhibited an increase in EC50, and a reduction in Vmax was observed in CHO-beta 2 but not beta 1 cells. CHW-beta 1 cells were resistant to acute agonist-mediated reduction in Vmax compared to CHW-beta 2 cells. More prolonged agonist exposure produced a modest reduction in Vmax and this effect was more noticeable when the CHW cells expressed lower levels of beta 1AR. To explore the role of protein kinases in these effects, digitonin-permeabilized CHW cells were loaded either with heparin (a beta ARK inhibitor) or a peptide inhibitor of PKA and exposed to agonist. In both beta 2AR- and beta 1AR-expressing cells, heparin inhibited the reduction in Vmax and the PKA inhibitor blocked the increase in EC50. Finally, exposing CHW cells expressing either subtype to a permeable cyclic AMP derivative caused an increase in EC50 similar to that observed in agonist-treated cells, but without any reduction in maximal activity. Our data suggest that whereas PKA-mediated desensitization is not subtype-specific, human beta 1AR is more resistant to beta ARK-mediated desensitization compared to the human beta 2AR.
Zhou
X M
XM
Membrane Biochemistry Section, National Institute of Neurological Disorders and Stroke, National Institute of Health, Bethesda, MD 20892, USA.
Pak
M
M
Wang
Z
Z
Fishman
P H
PH
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002463:Cell Membrane Permeability; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005347:Fibroblasts; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D018342:Receptors, Adrenergic, beta-1 / Q000096:biosynthesis / Q000502:physiology*; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
D047428:Protein Kinase Inhibitors; D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D006493:Heparin; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; D007545:Isoproterenol