title: Subtype-specific regulation of receptor internalization and recycling by the carboxyl-terminal domains of the human A1 and rat A3 adenosine receptors: consequences for agonist-stimulated translocation of arrestin3.
abstract: In this study, we have characterized the differential effects on inhibitory adenosine receptor (AR) trafficking of disrupting predicted sites for palmitoylation and phosphorylation within each receptor's carboxyl terminus. While a Cys(302,305)Ala-mutated rat A(3)AR mutant internalizes significantly faster than the wild-type (WT) receptor in response to agonist exposure, analogous mutation of the human A(1)AR (Cys(309)Ala) had no effect on receptor internalization. Moreover, unlike the WT A(3)AR, the entire pool of internalized mutant A(3)AR is able to recycle back to the plasma membrane following agonist removal. These properties do not reflect utilization of an alternative trafficking pathway, as internalized WT and mutant A(3)ARs both accumulate into transferrin receptor-positive endosomal compartments. However, receptor accumulation into endosomes is dependent upon prior G-protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptor's carboxyl terminus, as replacement of the carboxyl-terminal domain of the human A(1)AR with the 14 GRK-phosphorylated amino acids of the rat A(3)AR confers rapid agonist-mediated endosomal accumulation of the resulting chimeric A(1)CT3AR. Sensitivity to GRK-mediated phosphorylation also dictates the distinct redistribution of arrestin3 observed upon agonist exposure. Thus, while the nonphosphorylated A(1)AR redistributes arrestin3 from the cytoplasm to punctate clusters at the plasma membrane, GRK-phosphorylated WT and Cys(302,305)Ala-mutated A(3)ARs, as well as the A(1)CT3AR chimera, each induce the redistribution of arrestin3 into punctate accumulations both at the plasma membrane and within the cytoplasm. Neither the human A(1)AR nor the rat A(3)AR colocalized with arrestin3 under basal or agonist-stimulated conditions. Together, these results demonstrate that inhibitory AR-mediated changes in arrestin3 distribution are subtype-specific, with specificity correlating with the sensitivity of the receptor's carboxyl-terminal domain to GRK phosphorylation. In the case of the rat A(3)AR, sensitivity to GRK-mediated internalization appears to be regulated in part by the integrity of putative palmitate attachment sites upstream of its GRK phosphoacceptor sites.
authors.affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, UK.
authors.forename: Gail
authors.initials: G
authors.lastname: Ferguson
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Watterson
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Palmer
mesh_terms: D000409:Alanine / Q000235:genetics; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D003545:Cysteine / Q000235:genetics; D011992:Endosomes / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D058906:Purinergic P1 Receptor Agonists; D051381:Rats; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D019390:Arrestins; D008164:Luminescent Proteins; D058906:Purinergic P1 Receptor Agonists; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1; D011993:Recombinant Fusion Proteins; C082333:arrestin3; D049452:Green Fluorescent Proteins; D019308:Palmitic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D003545:Cysteine; D000409:Alanine