Chemokines are pro-inflammatory mediators for the recruitment of leukocytes from the circulation to the site of an infection or injury. Chemokine receptors (ChRs) are seven transmembrane (7-TM) helix- containing G protein-coupled receptors (GPCRs); many are effective and selective drug targets for the treatment of inflammatory and immune disorders. The mechanisms of action of ChRs are not yet defined, due largely to a lack of information on their high-resolution structures and dynamics. In this project we will develop novel technologies to overcome the major hurdles that previously precluded structural determination of integral membrane proteins: adequate expression and purification, stabilization in detergent solutions, and crystal growth. Our major goal is to elucidate the ChRs' crystal structures.
Our Specific Aims are to 1) optimize the production of homogeneous ChRs suitable for crystal growth, via a novel adenovirus-mediated mouse liver expression system; 2) solubilize, stabilize and purify the ChRs in detergent solutions, and adapt lipidic cubic phase-based electrophoresis for their detergent-free purification; 3) solve the crystal structures of one representative of each of the two major ChR classes (CXCR and CCR), and optimize a novel procedure for growing 3-D crystals from 2-D crystals in the lipidic cubic phase. This project is being conducted by a talented team of researchers with an excellent track record, including solving the crystal structures of two 7-TM receptors (sensory rhodopsin II & bovine rhodopsin). Achieving our Specific Aims will help elucidate the ChRs' mechanism of action, and lay the foundation for rational design of highly potent and selective new anti-inflammatory and immunomodulatory drugs targeting sites unique to specific receptor states. Health Relevance: While vital for proper wound healing and immune responses to fight infections, abnormal chemokine action also plays a role in such inflammatory diseases as arthritis and auto-immune disease as Lupus. Chemokines act via specific cell surface receptors which have been extremely difficult to characterize structurally. Our project seeks to overcome the obstacles to such characterization and so lay the foundation for developing drugs that could boost our immune system when we are faced with a life- threatening infection, or to dampen it to prevent tissue damage caused by autoimmune disorders (e.g. Lupus) or rejection of transplanted organs. ? ? ?
| Sarmiento, Jose; Shumate, Christie; Suetomi, Katsutoshi et al. (2011) Diverging mechanisms of activation of chemokine receptors revealed by novel chemokine agonists. PLoS One 6:e27967 |
| Joseph, Prem Raj B; Sarmiento, Jose M; Mishra, Anurag K et al. (2010) Probing the role of CXC motif in chemokine CXCL8 for high affinity binding and activation of CXCR1 and CXCR2 receptors. J Biol Chem 285:29262-9 |
| Sarmiento, J; Kypreos, K E; Prado, G N et al. (2009) Adenovirus mediated expression ""in vivo"" of the chemokine receptor CXCR1. J Struct Funct Genomics 10:17-23 |
| Vargas, Karina J; Sarmiento, José M; Ehrenfeld, Pamela et al. (2009) Postnatal expression of V2 vasopressin receptor splice variants in the rat cerebellum. Differentiation 77:377-85 |
