Structural and Functional Analysis of the Chemokine CCL27 and the Expression and Purification of Silent Chemokine Receptors D6 and DARC

Download or read book Structural and Functional Analysis of the Chemokine CCL27 and the Expression and Purification of Silent Chemokine Receptors D6 and DARC written by Ariane L. Jansma and published by . This book was released on 2009 with total page 206 pages. Available in PDF, EPUB and Kindle. Book excerpt: Chemokines are small chemoattractant proteins that function by binding to G-protein coupled receptors (GPCRs) on a wide variety of cell types, triggering cascades of intracellular signaling pathways. While they are best known for their role in leukocyte migration, both in standard immune surveillance and maintenance, as well as in response to inflammation, chemokines are involved in a variety of physiological and pathophysiological processes. In addition to their complex function, the chemokine network itself is highly complex, with some chemokines being specific to one receptor, while others activate multiple receptors expressed on different cell types and in some cases, resulting in very different cellular responses. This project in part involves the chemokine CCL27, which is expressed in skin and selectively chemoattracts CLA memory T cells expressing the chemokine receptor, CCR10. The first set of aims for this project involved a comprehensive analysis of the structural and functional mechanism contributing to the biological diversity of CCL27. This was accomplished through a biophysical characterization of the oligomerization properties, residues targeting receptor activation, as well as sites of glycosaminoglycan (GAG) interactions for this chemokine. The results suggest that CCL27 exists in multiple oligomeric states, and its unique oligomerization patterns appear to play a role in the diversity of its multiple binding partners. The second major project aim involves optimization of the expression and purification of the silent chemokine receptors D6 and the Duffy Antigen Receptor for Chemokines (DARC). D6 and DARC are termed silent receptors because they bind many chemokines with high affinity and specificity, but unlike other chemokine receptors, they do not signal through G-proteins. Instead, they act as regulators, either by targeting their chemokine ligands for degradation, or by shuttling them from one location to another. One of the rate-limiting steps in the biophysical characterization of 7-transmembrane helical chemokine receptors is obtaining sufficient amounts of purified, functional protein. In this project, a tetracycline-indicuble mammalian cell expression system is applied to both D6 and DARC. Results from the initial test expressions and purifications indicate that this method was successful in generating solubilized receptor protein.