title: Adenosine A(2A) receptor dynamics studied with the novel fluorescent agonist Alexa488-APEC.
abstract: G protein-coupled receptors, such as the adenosine A(2A) receptor, are dynamic proteins, which undergo agonist-dependent redistribution from the cell surface to intracellular membranous compartments, such as endosomes. In order to study the kinetics of adenosine A(2A) receptor redistribution in living cells, we synthesized a novel fluorescent agonist, Alexa488-APEC. Alexa488-APEC binds to adenosine A(2A) (K(i)=149+/-27 nM) as well as A(3) receptors (K(i)=240+/-160 nM) but not to adenosine A(1) receptors. Further, we characterized the dose-dependent increase in Alexa488-APEC-induced cAMP production as well as cAMP response element binding (CREB) protein phosphorylation, verifying the ligand's functionality at adenosine A(2A) but not A(2B) receptors. In live-cell imaging studies, Alexa488-APEC-induced adenosine A(2A) receptor internalization, which was blocked by the competitive reversible antagonist ZM 241385 and hyperosmolaric sucrose. Further, internalized adenosine A(2A) receptors co-localized with clathrin and Rab5, indicating that agonist stimulation promotes adenosine A(2A) receptor uptake through a clathrin-dependent mechanism to Rab5-positive endosomes. The basic characterization of Alexa488-APEC described here showed that it provides a useful tool for tracing adenosine A(2A) receptors in vitro.
authors.affiliation: Karolinska Institutet, Department of Physiology & Pharmacology, Sect. Receptor Biology & Signaling, S-171 77 Stockholm, Sweden.
authors.forename: Frank
authors.initials: F
authors.lastname: Brand
authors.forename: Athena M
authors.initials: AM
authors.lastname: Klutz
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Jacobson
authors.forename: Bertil B
authors.initials: BB
authors.lastname: Fredholm
authors.forename: Gunnar
authors.initials: G
authors.lastname: Schulte
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D058908:Adenosine A2 Receptor Agonists; D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000096:biosynthesis; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D011869:Radioligand Assay; D043705:Receptor, Adenosine A2A / Q000378:metabolism
chemical_list: D058908:Adenosine A2 Receptor Agonists; D017362:Cyclic AMP Response Element-Binding Protein; D005456:Fluorescent Dyes; D043705:Receptor, Adenosine A2A; C084956:N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine; D019830:Adenosine-5'-(N-ethylcarboxamide); D000242:Cyclic AMP; D048049:Extracellular Signal-Regulated MAP Kinases; D000241:Adenosine