2012年9月16日星期日

Modification of the beta 2-adrenergic receptor to engineer a receptor-effector complex for gene therapy.

Related Articles

Modification of the beta 2-adrenergic receptor to engineer a receptor-effector complex for gene therapy.

J Biol Chem. 2001 Aug 24;276(34):31596-601

Authors: Small KM, Brown KM, Forbes SL, Liggett SB

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 beta(2)-adrenergic receptor (beta(2)AR) 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 beta-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 beta(2)AR). This was fully restored by fusing the carboxyl terminus of the modified receptor to G alpha(s) (43.3 +/- 2.7 pmol/min/mg). The fully modified fused receptor was not activated by beta-agonists but rather by a nonbiogenic amine agonist that itself failed to activate the wild-type beta(2)AR. 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 beta(2)AR). Thus, despite extensive alterations in regions of conformational lability, the beta(2)AR 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.

PMID: 11402033 [PubMed - indexed for MEDLINE]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

没有评论:

发表评论