The 132-adrenergic receptor (132AR) plays a major role in the """"""""fight-or-flight"""""""" response including mediation of bronchodilation. Eliciting bronchodilation with agonists of the 132AR is, along with steroids to treat inflammation, a major treatment of chronic asthma. The effectiveness of the bronchodilators is known to decrease over time (desensitize), and as such the characterization of agonist desensitization of the 132AR has been the focus of a multitude of studies, and the evidence derived from these has made it a paradigm for the study of G protein coupled receptors (GPCRs). However, many questions remain concerning the molecular mechanisms of desensitization, and importantly. how the 132AR signaling complex resensitizes following removal of stimulation. At the systems biology level, there is a need for a dynamic modeling of the complex inhibitory feedback loops involving 132AR phosphorylation by PKA and multiple G protein coupled receptor kinase (GRK) subtypes, and their downstream sequelae such as arrestin binding, internalization, and activation of phosphodiesterase. Since most studies of 132AR desensitization have been performed with cell lines overexpressing the 132AR, there is a need for studies of desensitization in primary human cells expressing endogenous levels of the 132AR. Another aspect that has received little attention has been the development of inhibitors of desensitization. Our group has made significant inroads in ongoing studies of three areas of the 132AR desensitization process;characterization of GPCR activation of GRKs, development of a panel of inhibitors of GRK activity, and systems modeling of desensitization and resensitization, leading to the following specific aims: (1) characterization of 132AR desensitization in both HASM and model cell systems with a focus on quantitative systems modeling of the processes that control loss of both 132AR efficacy and downstream actions of the second messenger cAMP through phosphodiesterase hydrolysis;(2) determination of the mechanism of activation of GRKs by the 132AR receptor and the related GPCR rhodopsin through detailed structure/function studies of evolutionarily important GRK residues;and (3) development of peptide inhibitors that disrupt the GPCRlGRK interaction based on knowledge gained of important GRK and 132AR domains involved in the interaction.
One of the most profound and near universal aspects of GPCR regulation is that immediately following hormone or neurotransmitter activation, GPCR action is acutely desensitized, and with prolonged stimulation, receptors are further downregulated and degraded. The combination of acute and chronic desensitization and downregulation (hereafter referred to as desensitization) is crucial to maintenance of homeostasis following activation, and is intimately linked to cell function. From the standpoint of our focus on the 132AR, physiological relevance derives from consideration of the pleiotropic consequences of stimulation of the sympathetic nervous system that causes norepinephrine (NE) release at nerve endings, and epinephrine (EPI) and NE release (- 4:1 ratio) from the adrenal gland. Stimulation of the 132AR following hormone release plays a critical role in many important cell functions including relaxation of vascular and lung tracheal smooth muscle, and stimulation of heart contraction. Thus, pharmacological intervention in the physiological regulation of these events by the 132AR is of great importance. Numerous drugs that regulate 132AR function are currently in use, such as agonists for the treatment of asthma, COPD, and premature uterine contractions, and antagonists for the treatment of congestive heart failure. The currently approved major treatment of chronic severe asthma, the incidence of which in the United States is approximately 1 in 25, is the combination of a steroid to reduce inflammation, and a long acting 132AR agonist (LABA) for relaxation of tracheal and bronchiolar smooth muscle [11]. A key feature of chronic asthma is a dramatic shift of the normal balance of contraction and relaxation to sensitization of the contractile stimuli and desensitization of stimuli producing relaxation;however the biochemical changes in cells leading to asthma are poorly understood. There is considerable evidence that desensitization of 132AR action plays a major role [12-15], and as such it is of importance to understand the mechanisms of desensitization of 132AR which limit the potency and efficacy of 132AR agonist activity, and to develop novel inhibitors to block desensitization. While much is known about 132AR desensitization, work thus far with the human 132AR has almost invariably been accomplished in model systems, and a goal of the present work is to establish quantitatively the relevance of the major pathways of desensitization with endogenous receptor in human airway smooth muscle (HASM) cells in primary culture. Application of the inhibitors developed in this work will aid in both obtaining a better understanding of the roles of GRK- mediated desensitization and providing leads and directions for possible clinical use of the compounds.
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