Immunological identification of A2 adenosine receptors by two antipeptide antibody preparations

TM Palmer, KA Jacobson and GL Stiles

Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.

Two antipeptide antibody preparations were raised against deduced amino acid sequences within the presumed second extra-cellular loop (antibody TP/1) and the carboxyl-terminal domain (antibody TP/2) of the canine- derived A2 adenosine receptor (A2AR) cDNA species termed RDC8. Immunoblotting of canine liver plasma membranes with both TP/1 and TP/2 identified a single band of 52 kDa, which co-migrated with 125I-2-[4-[2- [2-[(4- azidophenyl)methylcarbonylamino]ethylaminocarbonyl]ethyl] phenyl]ethylamino-5'-N-ethylcarboxamidoadenosine-labeled receptor. However, in membranes prepared from canine striatum, photoaffinity labeling and immunoblotting with TP/2, but not TP/1, revealed a single band of 34 kDa; the identity of the band observed on the immunoblot as an A2AR was confirmed by the ability of TP/2 to specifically immunoprecipitate photoaffinity-labeled receptor from crude canine striatal membranes. The size difference between liver and striatal A2ARs was not due to tissue-specific proteolysis, because membranes from striatum were prepared with a protease inhibitor cocktail previously shown to be effective in inhibiting endogenous A2AR proteolysis during membrane preparation. Also, the protease-sensitive carboxyl-terminal region of the receptor had remained intact, because the peptide used to raise TP/2 antibodies resides in this domain of the molecule. The difference in size was also not due to a greater carbohydrate content of the liver receptor, because treatment of liver and striatal membranes with endoglycosidase F produced small mobility shifts for both receptors. Removal of N-linked carbohydrate chains also did not alter the inability of TP/1 to recognize the striatal A2AR. Hence, we conclude that the A2AR present in liver, which displays the predicted immunoreactivity of RDC8, is immunologically distinct from the A2AR expressed in striatum and that the latter may represent an additional A2AR subtype.

Volume 42, Issue 3, pp. 391-397, 09/01/1992
Copyright © 1992 by American Society for Pharmacology and Experimental Therapeutics

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