Engineering Chemokine Receptor Signaling Complexes In Vitro

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DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) represent the largest protein superfamily in humans, with nearly 1000 members. These receptors coordinate intercellular communication via the transduction of a wide range of stimuli involved in sensation, neurotransmission, development, emotion, cognition, and function in the CNS, endocrine and immune systems. Chemokine receptors are an important class of GPCRs that are best known for their pivotal role in immune surveillance, where they control the migration and activation of leukocytes in an effort to detect and resolve physiological abnormalities such as cancer and infection. However, inappropriate expression or regulation of these receptors is associated with an extraordinary number of pathologies including inflammatory diseases, cancer and AIDS; thus there is significant interest in developing small molecule receptor antagonists that block the function of specific chemokine receptors. To this end, our long-term goal is to obtain structural information that can aid the drug discovery process. We are also interested in delineating fundamental information about chemokine receptor structures, dynamics and conformational changes associated with agonist and antagonist induced states, molecular interactions that stabilize the corresponding active and inactive states of the receptors, and ultimately the relationship between these properties and signaling output. However limited structural, biophysical and mechanistic information is available for membrane proteins. GPCRs are particularly difficult eukaryotic membrane proteins as evidenced by the fact that only two structures, bovine rhodopsin and the 22-adrenergic receptor, have been solved. The lack of structures is due to two main obstacles: protein expression and functional reconstitution in artificial membranes. However, we have succeeded in expressing the chemokine receptor, CCR1, at a level sufficient for biophysical studies. The aims of this project are to: 1) Further optimize the expression and purification of CCR1, 2) Optimize conditions for generating homogeneous preparations of functional CCR1 in artificial membranes, 3) Characterize in vivo, the pharmacological requirements for producing CCR1 in a high affinity functional state, 4) Engineer ternary signaling complexes in vitro, involving CCR1, chemokine ligands and heterotrimeric G proteins and 5) Explore alternative heterologous systems for chemokine receptor expression. In the course of these studies, many important pharmacological issues will be addressed such as the precise requirement of G proteins for receptor function, and sequence motifs involved in receptor trafficking. We will lay the foundation for future structural and biophysical studies that can be interpreted in the context of a well-characterized biological system. Significance: Our studies will have broad impact on biophysical studies of chemokine receptors and GPCRs in general. Given that approximately 50% of marketed drugs target GPCRs, impact may eventually extend to drug design where chemokine receptors have become important therapeutic targets. PUBLIC HEALTH RELEVANCE: Under normal physiological conditions, chemokines and their receptors are involved in processes centered around their ability to control cell migration in the context of immune system function and development. However, inappropriate chemokine-mediated cell migration and inflammation causes or contributes to the pathology of many diseases such as asthma, rheumatoid arthritis, multiple sclerosis, heart disease, and cancer. Understanding the molecular details of chemokine:receptor interactions and function, may facilitate the design of small molecule and protein therapeutics for many diseases.