G protein-coupled regulate many critical physiological processes. Two prototypical members of this family, the beta2-adrenergic receptor (beta2AR) and substance P receptor are the key mediators of the cardiovascular system and of pain transmission in the peripheral and central nervous systems, respectively. Although each of these receptors responds to a different ligand, they share common structural features. Moreover, the desensitization and resensitization of their signaling abilities and their ability to internalize are regulated by G protein- coupled receptor kinases (GRKS) and beta arrestins. Almost all of the information concerning the regulation of GPCRs by the GRKs and beta- arrestins derives from studies using either cellular membrane extracts or reconstituted system of proteins. The overall objective of the proposed research is to define the molecular mechanisms governing GPCR signaling by studying these processes in live cells, with the ultimate goal of understanding the cell physiology and pathophysiology arising from abnormal GPCR regulation. The beta2-adrenergic receptor and substance P receptor will be used as representative receptors, since their overall behavior should reflect that of many other GPCR family members. Assays based upon the ability to visualize the behavior of the beta2AR or the substance P receptor, GRK2, and beta-arrestin 1 and 2, utilizing their fluorescent chimeras, will be employed to follow receptor desensitization and cell redistribution in real-time.
Aim I -is to study the agonist-mediated desensitization of signaling in the beta2- adrenergic and substance P receptors in real-time in living cells using novel green fluorescent protein variants of GRKs and beta arrestins. The results should provide significant insight into the molecular regulation of GPCRs in complex cellular systems.
Aim II - is to study the cellular endocytic pathway responsible for beta2-adrenergic receptor and substance P receptor down regulation. The results will provide a basis to understand how GPCRs are targeted for degradation or recycling as a result of prolonged agonist exposure. The proposed studies should enhance our understanding of the fundamental molecular and cellula properties that are important for maintenance of normal GPCR signal transduction. Insight gained from them should provide a foundation for the development of new treatment regiments that target diseases such as hypertension, heart failure, and chronic pain syndromes.
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