Antibodies for the Immunochemistry of the Human β3‐Adrenergic Receptor

• Published in: European journal of biochemistry Vol. 224; no. 2; pp. 761 - 770
• Main Authors: Guillaume, Jean‐Luc, Petitjean, Françoise, Haasemann, Martina, Bianchi, Cesario, Eshdat, Yuval, Strosberg, A. Donny
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-09-1994
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1994.00761.x

Based on the amino acid sequence deduced from the recently cloned human β3‐adrenergic receptor (huβ3AR) gene, polyclonal antibodies were prepared against synthetic peptides, corresponding to regions of huβ3AR 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 β1 and β2 adrenergic receptors. Affinity‐purified antibodies directed against N‐terminal, extracellular or intracellular loops and C‐terminal peptides reacted specifically with the huβ3AR and not with either the human β1 or β2 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 huβ3AR gene; in addition, the presence of the receptor protein was established in a human tissue (gall bladder). Immuno‐affinity chromatography of solubilized CHO huβ3AR‐containing cell membranes allowed the isolation of huβ3AR 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.