Modification of the β2-Adrenergic Receptor to Engineer a Receptor-Effector Complex for Gene Therapy

Abstract

Depressed G-protein-coupled receptor (GPCR) signaling has been implicated as a component of the pathophysiology of a number of complex diseases including heart failure and asthma, and augmentation or restoration of signaling by various means has been shown to improve organ function. Because some properties of native GPCRs are disadvantageous for ectopic therapeutic expression, we utilized the β2-adrenergic receptor (β2AR) as a scaffold to construct a highly modified therapeutic receptor-effector complex (TREC) suitable for gene therapy. Altogether, 19 modifications were made to the receptor. The ligand-binding site was re-engineered in TM-3 so that a β-hydroxylmethyl side chain acts as a proton donor for the binding of a novel ligand. In addition, sites critical for agonist-promoted down-regulation in the amino terminus and for phosphorylation by GPCR kinases, and protein kinases A and C, in the third intracellular loop and the carboxyl terminus of the receptor were altered. These modifications of the receptor resulted in depressed agonist-stimulated adenylyl cyclase activity (26.8 ± 2.1 versus 41.4 ± 8 pmol/min/mg for wild-type β2AR). This was fully restored by fusing the caroxyl terminus of the modified receptor to Gαs (43.3 ± 2.7 pmol/min/mg). The fully modified fused receptor was not activated by β-agonists but rather by a nonbiogenic amine agonist that itself failed to activate the wild-type β2AR. This two-way selectivity thus provides targeted activation based on physiologic status. Furthermore, the TREC did not display tachyphylaxis to prolonged agonist exposure (desensitization was 1 ± 5% versus 55 ± 4% for wild-type β2AR). Thus, despite extensive alterations in regions of conformational lability, the β2AR can be tailored to have optimal signaling characteristics for gene therapy. As a general paradigm, TRECs for enhancement of other G-protein signaling appear to be feasible for modification of other pathologic states.