title: Glycoprotein nature of the A2-adenosine receptor binding subunit.
abstract: Mammalian A2-adenosine receptor binding subunits (A2AR) can be visualized by covalent labeling with the photoaffinity crosslinking ligand 125I-2-[4-[2-[2-[(4-aminophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl]phenyl]ethylamino-5'-N-ethylcarboxamidoad enosine or directly with the azide derivative described in this paper. The protein comprising the A2-adenosine receptor binding subunit migrates with a Mr of 45,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, the glycoproteins representing the radiolabeled A1- and A2-adenosine receptor binding subunit from bovine brain were compared by partial peptide maps and following treatment with exo- and endoglycosidases. Peptide maps using two separate proteases reveal that the A1- and A2-adenosine receptor binding subunits share no common peptide fragments by two-dimensional gel electrophoresis. Endoglycosidase F treatment of labeled A2AR results in a single labeled peptide of Mr 38,000 without intermediate peptides, suggesting a single N-linked carbohydrate chain. The labeled A2AR demonstrates a sensitivity to neuraminidase, as evidenced by an increased mobility on gel electrophoresis, suggesting the receptors contain a glycan component containing terminal sialic acid. Treatment of the labeled A2AR with alpha-mannosidase reveals two distinct populations of A2ARs, one of which is sensitive and the other resistant to the enzyme. The nonadditivity of sequential treatments with the two exoglycosidases suggests, a heterogeneous population of A2AR containing either complex- or high mannose-type carbohydrate chains. These data suggest the A2AR is a Mr 45,000 glycoprotein with a single carbohydrate chain of either the complex or high mannose type. In addition, the A1- and A2ARs are distinct glycoproteins, as evidenced by their differing molecular weights (before and after deglycosylation) and distinct peptide maps.
authors.affiliation: Department of Medicine, Cardiology, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: W W
authors.initials: WW
authors.lastname: Barrington
authors.forename: K A
authors.initials: KA
authors.lastname: Jacobson
authors.forename: G L
authors.initials: GL
authors.lastname: Stiles
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism; D002417:Cattle; D003342:Corpus Striatum / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis*; D006026:Glycoside Hydrolases; D007462:Iodobenzenes; D010449:Peptide Mapping; D011983:Receptors, Purinergic / Q000032:analysis*
chemical_list: D000345:Affinity Labels; D001386:Azides; D006023:Glycoproteins; D007462:Iodobenzenes; D011983:Receptors, Purinergic; C060851:2-(4-(2-(2-((4-aminophenyl)methylcarbonylamino)ethylaminocarbonyl)ethyl)phenyl)ethylamino-5'-N-ethylcarboxamidoadenosine; C065390:2-(4-(2-(2-((4-azidophenyl)methylcarbonylamino)ethylaminocarbonyl)ethyl)phenyl)ethylamino-5'-N-ethylcarboxamidoadenosine; D006026:Glycoside Hydrolases; D000241:Adenosine