Cargo-mediated regulation of a rapid Rab4-dependent recycling pathway.
Membrane trafficking is well known to regulate receptor-mediated signaling processes, but less is known about whether signaling receptors conversely regulate the membrane trafficking machinery. We investigated this question by focusing on the beta-2 adrenergic receptor (B2AR), a G protein-coupled receptor whose cellular signaling activity is controlled by ligand-induced endocytosis followed by recycling. We used total internal reflection fluorescence microscopy (TIR-FM) and tagging with a pH-sensitive GFP variant to image discrete membrane trafficking events mediating B2AR endo- and exocytosis. Within several minutes after initiating rapid endocytosis of B2ARs by the adrenergic agonist isoproterenol, we observed bright "puffs" of locally increased surface fluorescence intensity representing discrete Rab4-dependent recycling events. These events reached a constant frequency in the continuous presence of isoproterenol, and agonist removal produced a rapid (observed within 1 min) and pronounced (approximately twofold) increase in recycling event frequency. This regulation required receptor signaling via the cAMP-dependent protein kinase (PKA) and a specific PKA consensus site located in the carboxyl-terminal cytoplasmic tail of the B2AR itself. B2AR-mediated regulation was not restricted to this membrane cargo, however, as transferrin receptors packaged in the same population of recycling vesicles were similarly affected. In contrast, net recycling measured over a longer time interval (10 to 30 min) was not detectably regulated by B2AR signaling. These results identify rapid regulation of a specific recycling pathway by a signaling receptor cargo.
Department of Psychiatry, University of California San Francisco, San Francisco, CA 94158, USA.
Guillermo A
GA
Yudowski
Manojkumar A
MA
Puthenveedu
Anastasia G
AG
Henry
Mark
M
von Zastrow
D058672:Adrenergic beta-2 Receptor Antagonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D005089:Exocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007545:Isoproterenol / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism; D011990:Receptors, Transferrin / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors; D020695:rab4 GTP-Binding Proteins / Q000378:metabolism*
D058672:Adrenergic beta-2 Receptor Antagonists; D000318:Adrenergic beta-Agonists; D018343:Receptors, Adrenergic, beta-2; D011990:Receptors, Transferrin; D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D020695:rab4 GTP-Binding Proteins; D007545:Isoproterenol