Antibodies for the Immunochemistry of the Human β3‐Adrenergic Receptor

Abstract

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.

The tissue distribution of the human β3-adrenoceptor studied using a monoclonal antibody: Direct evidence of the β3-adrenoceptor in human adipose tissue, atrium and skeletal muscle

“…Possibly, this re¯ects clustering of b 3 -adrenoceptors in sympathetically innervated cells. Furthermore, it is noteworthy that the staining pattern obtained in gall bladder was quite different from that reported in a previous study 23 using a polyclonal anti-peptide antibody. In the latter report, staining was associated primarily with arteriolar vascular smooth muscle cells, with negligible staining in the lamina propria.…”

“…Data describing the immunohistochemical localisation of the b 3 -adrenoceptor has been limited to a single study using an anti-peptide polyclonal antibody, 23 which detected the receptor only in human gall bladder, a tissue that exhibits a relatively high level of b 3 -adrenoceptor mRNA. 17,19 The relative lack of sensitivity of this approach may be due to the dif®culty of producing a suitable high af®nity antibody using a short polypeptide immunogen.…”

Cellular Trafficking of Human α1a-Adrenergic Receptors Is Continuous and Primarily Agonist-Independent

“…Two studies also examined localization of ␣ 1 ARs in native human smooth muscle cells and found internal as well as membrane-bound populations of ␣ 1a AR (Hrometz et al, 1999;Mackenzie et al, 2000). This is similar to the situation for the ␣ 2 ARs, where ␣ 2A -and ␣ 2B ARs are predominantly surface localized, whereas the ␣ 2C AR is predominantly intracellular (von Zastrow et al, 1993;Wozniak and Limbird, 1996;Daunt et al, 1997) but contrasts with ␤ARs, which are surface localized (Guillaume et al, 1994;Barak et al, 1997;McLean and Milligan, 2000).…”

Involvement of β 3 -adrenergic receptor activation via cyclic GMP- but not NO-dependent mechanisms in human corpus cavernosum function

“…The ␤ 3 -receptor plays a major role in lipolysis but the role and distribution of ␤ 3 -receptors in other specific sites have not been extensively studied. Tissue distribution studies have demonstrated the presence of ␤ 3 -receptors not only in adipose tissues but also in human gall bladder and more recently in colon, prostate, and skeletal muscle (9)(10)(11). In addition, ␤ 3 -receptor stimulation has been recently demonstrated to elicit vasorelaxation of rat aorta through activation of NO synthase and the subsequent increase in tissue levels of cGMP (12).…”