Beta-arrestin scaffolding of phosphatidylinositol 4-phosphate 5-kinase Ialpha promotes agonist-stimulated sequestration of the beta2-adrenergic receptor.
Members of the seven-transmembrane receptor (7TMR) superfamily are sequestered from the plasma membrane following stimulation both to limit cellular responses as well as to initiate novel G protein-independent signaling pathways. The best studied mechanism for 7TMR internalization is via clathrin-coated pits, where clathrin and adaptor protein complex 2 nucleate and polymerize upon encountering the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) to form the outer layer of the clathrin-coated vesicle. Activated receptors are recruited to clathrin-coated pits by beta-arrestins, scaffolding proteins that interact with agonist-occupied 7TMRs as well as adaptor protein complex 2 and clathrin. We report here that following stimulation of the beta2-adrenergic receptor (beta2-AR), a prototypical 7TMR, beta-arrestins bind phosphatidylinositol 4-phosphate 5-kinase (PIP5K) Ialpha, a PIP(2)-producing enzyme. Furthermore, beta-arrestin2 is required to form a complex with PIP5K Ialpha and agonist-occupied beta2-AR, and beta-arrestins synergize with the kinase to produce PIP(2) in response to isoproterenol stimulation. Interestingly, beta-arrestins themselves bind PIP(2), and a beta-arrestin mutant deficient in PIP(2) binding no longer internalizes 7TMRs, fails to interact with PIP5K Ialpha, and is not associated with PIP kinase activity assayed in vitro. However, a chimeric protein in which the core kinase domain of PIP5K Ialpha has been fused to the same beta-arrestin mutant rescues internalization of beta2-ARs. Collectively, these data support a model in which beta-arrestins direct the localization of PIP5K Ialpha and PIP(2) production to agonist-activated 7TMRs, thereby regulating receptor internalization.
Howard Hughes Medical Institute, Departments of Medicine, Immunology, and Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Christopher D
CD
Nelson
Jeffery J
JJ
Kovacs
Kelly N
KN
Nobles
Erin J
EJ
Whalen
Robert J
RJ
Lefkowitz
D000818:Animals; D019390:Arrestins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000737:chemistry; D004705:Endocytosis; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D000071557:beta-Arrestins
D019390:Arrestins; D002966:Clathrin; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D000071557:beta-Arrestins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase