125I-2-[4-[2-[2-[(4-azidophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl]phenyl] ethylamino-5'-N-ethylcarboxamidoadenosine labels transmembrane span V of the A2a adenosine receptor.
We have shown previously that 125I-2-[4-[2-[2-[(4-azidophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl]phenyl] ethylamino-5'-N-ethylcarboxamidoadenosine (125I-azido-PAPA-APEC) specifically and selectively photolabels RDC8 A2a adenosine receptors that have been overexpressed in COS M6 cells. Glycosylated, 125I-azido-PAPA-APEC-labeled, wild-type (412 residues; 45,031 Da) and carboxyl-terminally truncated (315 residues; 35,427 Da) receptors migrate with apparent molecular masses of > 40 and 31.5 kDa, respectively, whereas unglycosylated or deglycosylated wild-type and truncated A2a receptors migrate with apparent molecular masses of 40 and 28.5 kDa, respectively. Because nonspecific photoincorporation is not a complication, the present peptide mapping studies of the full length and truncated canine A2a adenosine receptors were carried out on unpurified COS M6 membrane preparations. After partial proteolysis it became clear that glycosylation increased the apparent molecular mass of either the wild-type or mutant A2a receptor by approximately 3 kDa. Although the A2a receptor was readily cleaved by a variety of chemical reagents and proteases, trypsin and endoproteinase Glu-C generated the most reproducible and, in the case of trypsin, the most complete fragmentation patterns. Radiolabeled peptides were identified by their apparent molecular masses, (in)abilities to be recognized by an antipeptide antibody to amino acids Tyr155-Val172 of the presumed second extracellular loop of the receptor, and (in)sensitivities to endoglycosidase F and tunicamycin treatments. A prominent, 7-kDa, radiolabeled peptide that was generated by trypsin digestion implicated putative alpha-helix V in the binding of 125I-azido-PAPA-APEC.
Piersen
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CE
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6600.
True
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CD
Wells
J N
JN
D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry; D000345:Affinity Labels / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D004285:Dogs; D015180:Electrophoresis, Gel, Two-Dimensional; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010777:Photochemistry; D018047:Receptors, Purinergic P1 / Q000737:chemistry*; D012697:Serine Endopeptidases; D014357:Trypsin
D000345:Affinity Labels; D001386:Azides; D007457:Iodine Radioisotopes; D018047:Receptors, Purinergic P1; C065390:2-(4-(2-(2-((4-azidophenyl)methylcarbonylamino)ethylaminocarbonyl)ethyl)phenyl)ethylamino-5'-N-ethylcarboxamidoadenosine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D000241:Adenosine