Functional activity and regulation of human β2-adrenergic receptors expressed in Xenopus oocytes

The recently cloned human β-adrenergic cDNA and several mutated forms have been expressed in Xenopus laevis oocytes by injection of RNA made from the cDNA under the control of the bacteriophage SP6 promoter. The cDNA and gene of the β2-adrenergic receptor possess the unusual feature of having a second upstream ATG (-101 base pairs) and a 19-codon open reading frame 5' to the initiator methionine codon of the receptor. RNA lacking this upstream AUG and open reading frame was translated ~10-fold more efficiently both in an in vitro rabbit reticulocyte system and in oocytes. Injected oocytes but not water injected controls expressed typical β2-adrenergic receptors as asessed by ligand binding (450 fmol/mg membrane protein) and catecholamine-stimulated adenylate cyclase (~20 fold). Moreover, these receptors displayed typical agonist-induced homologous desensitization when oocytes were incubated with isoproterenol at room temperature for 3-24 h. Among a series of mutations, truncations of the membrane-anchored core of the receptor eliminated receptor binding and cyclase stimulating activity. In contrast, disruption of one of the cAMP-dependent protein kinase phosphorylation sites or removal of the serine/threonine-rich carboxyl terminus had little or no effect on these functions or on the extent of agonist-induced desensitization relative to that observed with native receptor. These studies validate the β2-adrenergic nature of the cloned human β-adrenergic cDNA, document the utility of the Xenopus oocyte system for studying functional and regulatory properties of receptors coupled to adenylate cyclase, and suggest the possibility that elements in the 5' untranslated region of the β2-adrenergic receptor RNA may regulate its translation in vivo.

Duke Scholars

Author Robert J. Lefkowitz Medicine, Cardiology