Chemokines are critical mediators of cell migration during immune surveillance, lymphocyte development, and inflammation. They function by binding to seven transmembrane G-protein coupled receptors (GPCRs), causing conformational changes that trigger intracellular signaling pathways involved in cell movement and receptor activation. Although chemokines were designed to carry out developmental and protective roles, many diseases result from inappropriate expression, regulation, or utilization of these proteins. Specific chemokine receptors provide the portals for HIV to get into cells, while others contribute to inflammatory disease, and migration (metastasis) of many types of cancers. Thus, there is considerable interest in delineating the structural details of how these proteins function, and mechanisms for antagonizing their function. Presently there is a fair amount of information on the structure and receptor binding epitopes of the ligands, which are small (8-12 kDa) soluble proteins. By contrast, very little structural information is known about their G protein-coupled receptors. This lack of information is due to the inherent difficulties in studying membrane proteins, particularly those of eukaryotic origin. The first obstacle in characterizing GPCRs has been the inability to obtain sufficient levels of protein because membrane proteins are usually toxic when over- expressed. Structural characterization of GPCRs by crystallography or NMR is also significantly more difficult than for soluble proteins. However, after more than a decade of structure-function studies of ligands, we recently began focusing on the receptors and have two receptors (D6 and CCR1) at levels sufficient for biophysical studies. In this proposal, we plan to develop and apply Hydrogen Deuterium Exchange coupled with Mass spectrometry (DXMS) to characterize the binding interfaces between chemokines and their receptors.
In Aim 1, we will optimize the functional reconstitution of purified receptor.
In Aim 2, we will define the receptor-binding surface on the chemokine ligand(s).
In Aim 3, we will develop methods to define the chemokine-binding surface on the receptors. In addition to contact sites, it should be possible to identify regions of the intracellular loops of the receptor that are involved in activation of downstream signaling molecules like G proteins, which undergo changes in stability or conformation. If successful, the methods should have broad applicability for characterizing chemokine:receptor interactions. For example, the results of these studies can be used to focus and complement mutagenesis studies. Since there are many different ligands of CCR1 and D6, different complexes can ultimately be investigated; comparison of agonists and antagonists, especially with respect to changes in intracellular loops, will be particularly interesting.
These specific aims will be accomplished by the synergistic activities of two groups: Dr. Handel, an expert in the structural biology of chemokines and receptors, and Dr. Woods, an expert in DXMS methodology, who is particularly interested, and already involved, in developing DXMS methods for membrane proteins.
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. Developing methods like DXMS to understand the molecular details of chemokine:receptor interactions and function, may facilitate the design of small molecule and protein therapeutics for many diseases. ? ? ?
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