L J Emorine

Molecular characterization of the mouse beta 3-adrenergic receptor: relationship with the atypical receptor of adipocytes.

The gene encoding the murine beta 3‐adrenergic receptor (beta 3AR) has been isolated. It translates into a polypeptide of 388 amino acid residues which shows 82% overall homology with the human beta 3AR. In Southern blot experiments, a probe derived from the murine beta 3AR gene hybridizes to a unique restriction fragment in the murine and human genomes. In both species, the beta 3AR gene is located on chromosome 8, in regions (8A2‐‐‐‐8A4 in mouse, and 8p11‐‐‐‐8p12 in man) which are conserved between mouse and man. The pharmacological profile of the mouse beta 3AR strongly resembles that of the human beta 3AR. It is characterized by a low affinity toward the radiolabelled beta‐adrenergic antagonist [125I]Iodocyanopindolol and a low efficiency of other antagonists such as propranolol, ICI 118551 or CGP 20712A to inhibit cAMP production induced by isoproterenol. Another salient feature shared by the murine and the human beta 3ARs is the very potent effect of the lipolytic compound BRL 37344 on cAMP accumulation and the partial agonistic effect of the beta 1‐ and beta 2‐adrenergic antagonists CGP 12177A, oxprenolol and pindolol. These properties are very close to those ascribed to the atypical beta AR of rodent adipocytes. In addition, Northern blot analyses indicate that the beta 3AR gene is mainly expressed in mouse brown and white adipose tissues, suggesting that the murine beta 3AR described here is the atypical beta AR involved in the control of energy expenditure in fat tissue.

Selective binding of ligands to beta 1, beta 2 or chimeric beta 1/beta 2-adrenergic receptors involves multiple subsites.

The molecular basis of ligand binding selectivity to beta‐adrenergic receptor subtypes was investigated by designing chimeric beta 1/beta 2‐adrenergic receptors. These molecules consisted of a set of reciprocal constructions, obtained by the exchange between the wild‐type receptor genes of one to three unmodified transmembrane regions, together with their extracellular flanking regions. Eight different chimeras were expressed in Escherichia coli and studied with selective beta‐adrenergic ligands. The evaluation of the relative effect of each chimeric exchange on ligand binding affinity was based on the analysis of delta G values, calculated from the equilibrium binding constants, as a function of the number of substituted beta 2‐adrenergic receptor transmembrane domains. The data showed that the contribution of each exchanged region to subtype selectivity varies with each ligand; moreover, while several regions are critical for the pharmacological selectivity of all ligands, others are involved in the selectivity of only some compounds. The selectivity displayed by beta‐adrenergic compounds towards beta 1 or beta 2 receptor subtypes thus results from a particular combination of interactions between each ligand and each of the subsites, variably distributed over the seven transmembrane regions of the receptor; these subsites are presumably defined by the individual structural properties of the ligands.

Idiotypy of catecholamine-binding proteins

The idiotypic and antiidiotypic response to alprenolol, a beta-adrenergic antagonist, was studied both in rabbits and in mice. Rabbit polyclonal anti-alprenolol antibodies showed binding properties for catecholamine analogs, agonists as well as antagonists, similar to those of the beta-adrenergic receptors. The variability of the anti-alprenolol response was studied by using mouse monoclonal antibodies specific for alprenolol. While the binding properties showed great variations in affinity, the response seemed restricted to the heavy chain classes gamma 1 and gamma 2a. N-terminal sequencing of the light and heavy chains and restriction maps of the corresponding genes suggest that the antibodies use particular subgroups infrequently found in antibodies specific for other antigens. The cyclical antiidiotypic response in rabbits immunized with polyclonal antibodies and in mice immunized with monoclonal antibodies were compared. The response of the latter was dependent on the choice of the monoclonal antibody used to elicit the antiidiotypic response. Finally, the agonist-like properties of a monoclonal antiidiotypic antibody directed against one of the monoclonal anti-alprenolol antibodies were studied extensively. The ability to recognize beta-adrenergic receptors was documented by Western blot and direct immunoprecipitation and visualized by immunofluorescence. The antiidiotypic antibody stimulated catecholamine-sensitive adenylate cyclase and this effect was blocked by the beta-adrenergic antagonist propranolol.