Leukocytes play an important role in host defense against invading microorganisms through their ability to generate superoxide radicals and release degradative enzymes following migration to sites of inflammation. However, these same responses can also participate in the formation of numerous pathological conditions such as autoimmune diseases and tissue injuries. The goal of this work is to understand the molecular mechanisms involved in the regulation of leukocyte activation initiated by chemoattractant receptors.
The specific aims of this proposal include the determination of the involvement of G protein coupled receptor kinases in the phosphorylation and subsequent desensitization of the N- formyl peptide receptor (FPR) and in particular the identification of the structural basis for the interaction between the G protein coupled receptor Beta ARK, with the carboxy terminus of the FPR. This work will also examine the role of phosphorylation of the FPR in the desensitization of other chemoattractant receptors and the involvement of additional factors in this process. As a model, this work will utilize the cloned human FPR cDNA expressed exogenously in mammalian cell lines, such as the promyelocytic line, HL60. In addition, the carboxy terminus of the FPR will be expressed as a fusion protein allowing its isolation in large quantities and its detailed characterization in vitro with highly purified Beta ARK. Use of mutant fusion protein constructs is expected to identify regions of the FPR phosphorylated by Beta ARK and regions involved in the activation of Beta ARK. The introduction of these mutations into the FPR expressed in its native environment will reveal mechanisms involved in the desensitization of chemoattractant receptors. In addition, the effects of these mutations on the binding of ligand and the activation of G proteins will be assessed. Information obtained from the proposed studies is expected to extend our knowledge of the regulation of signal transduction by chemoattractant and other stimulatory receptors, and to provide a basis for the development of potential therapeutic means of intervening in chronic cellular activation.
| Wagener, Brant M; Marjon, Nicole A; Revankar, Chetana M et al. (2009) Adaptor protein-2 interaction with arrestin regulates GPCR recycling and apoptosis. Traffic 10:1286-300 |
