Antibodies for the immunochemistry of the human beta 3-adrenergic receptor.
Based on the amino acid sequence deduced from the recently cloned human beta 3-adrenergic receptor (hu beta 3AR) gene, polyclonal antibodies were prepared against synthetic peptides, corresponding to regions of hu beta 3AR 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 beta 1 and beta 2 adrenergic receptors. Affinity-purified antibodies directed against N-terminal, extracellular or intracellular loops and C-terminal peptides reacted specifically with the hu beta 3AR and not with either the human beta 1 or beta 2 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 hu beta 3AR gene; in addition, the presence of the receptor protein was established in a human tissue (gall bladder). Immuno-affinity chromatography of solubilized CHO hu beta 3AR-containing cell membranes allowed the isolation of hu beta 3AR 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.
Guillaume
J L
JL
Institut Cochin de Génétique Moléculaire, Laboratoire d'Immuno-Pharmacologie Moléculaire, Paris, France.
Petitjean
F
F
Haasemann
M
M
Bianchi
C
C
Eshdat
Y
Y
Strosberg
A D
AD
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011943:Receptors, Adrenergic, beta / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D022702:Receptors, Adrenergic, beta-3; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D014162:Transfection
D000906:Antibodies; D010455:Peptides; D011943:Receptors, Adrenergic, beta; D022702:Receptors, Adrenergic, beta-3; D011994:Recombinant Proteins