Based on the amino acid sequence deduced from the recently cloned human P3-adrenergic receptor (hup3AR) gene, polyclonal antibodies were prepared against synthetic peptides, corresponding to regions of huP3AR presumed to be exposed at the outer or the inner side of the membrane on the basis of the putative three-dimensional structure of the previously characterized /31 and P2 adrenergic receptors. Affinity-purified antibodies directed against N-terminal, extracellular or intracellular loops and C-terminal peptides reacted specifically with the hup3AR and not with either the human Pl or p2 adrenergic receptor. Using these antibodies, it was demonstrated that the receptor is present at the surface of Chinese Hamster Ovary (CHO) cells transfected with the huP3AR gene; in addition, the presence of the receptor protein was established in a human tissue (gall bladder). Immuno-affinity chromatography of solubilized CHO hup3AR-containing cell membranes allowed the isolation of hup3AR protein with an overall yield of 30%. The degree of purity of the receptor was more than 80%, as assessed by N-terminal sequencing of the protein eluted from the column. Sequence analysis demonstrated the absence of a methionine residue at the N-terminal position, and suggested that the side chain of the asparagine residue at position 7 is glycosylated.
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