Chemokines, or chemoattractant cytokines, are important signaling molecules that regulate trafficking of immune cells during homeostatic processes or during a response to infection or inflammation. The chemokine CCL21 normally functions to recruit antigen presenting dendritic cells and naive T-cells expressing the chemokine receptor CCR7 to the lymph nodes. Subsequent antigen presentation results in activated T-cells, a key step in allowing the adaptive immune system to fight disease, and ultimately leads to activated macrophages, antibody producing B-cells and killer T-cells. Chemokines are also involved in numerous disease states. CCL21 targets metastatic cancer cells to the lymph nodes, a common site for formation of metastases. The presence of metastases in sentinel lymph nodes is used to stage the severity of numerous cancers. Structural studies on other chemokines, including RANTES/CCL5, MCP-1/CCL2, and SDF- 1/CXCL12, led to variants that function as antagonists or partial agonists that can reduce or eliminate cellular migration towards the wild-type chemokine. Also, small, drug-like ligands for CXCL12 can prevent activation of CXCR4. However, similar structural studies on CCL21 are lacking. The structure of CCL21 along with the oligmer state, will be determined in Aim 1. CCL21 activates the chemokine receptor CCR7. CCR7 is a heptahelical integral membrane G-protein coupled receptor (GPCR). Chemokines activate their receptors via a """"""""two state/ two site"""""""" mechanism by first binding to the receptor N-terminus (Site 1) and subsequently the chemokine N-terminus binds to a second site on the receptor (Site 2) causing high affinity binding and activation. Although recent progress in producing GPCRs for structural studies is exciting, we will investigate the interaction of CCL21 with the N-terminus of CCR7, thus mimicking the Site 1 interaction. Chemokine receptor N-termini contain post-translationally modified sulfotyrosine amino acids that increase affinity for chemokine. Recent studies suggest sulfotyrosine binding sites can be targeted with small molecule inhibitors.
Aim 2 seeks to identify the CCL21 binding site for N-terminus of CCR7 and the CCR7 sulfotyrosines. Subsequently, these binding sites will serve as targets for the development of CCL21 inhibitors with collaborators at R01 institutions. CCL21 also binds directly the N-terminus P-selectin glycoligand-1 (PSGL-1), an interaction that enhances chemotaxis of leukocytes.
Aim 3 will test the hypothesis that sulfotyrosines in the PSGL-1 N-terminus contribute to CCL21 binding, and, if sulfotyrosines are important for binding, will identify the PSGL-1 sulfotyrosine binding sites on CCL21. This proposal seeks to solve the structure of CCL21 while characterizing how CCL21 recognizes physiological significant binding partners. Information on these binding events will directly influence drug discovery studies that target chemokines, as this knowledge will be used by researchers at R01 institutions in their sulfotyrosine binding site directed drug screening efforts.
Chemokines, like CCL21, function as regulators of the immune system though trafficking of immune cells. However, CCL21 also recruits metastatic cancer cells to the lymph nodes where metastases commonly form. Cancers that have metastasized or spread are difficult to treat. An increased understanding of CCL21 is sought through structural studies that may help identify ways to reduce cancer metastasis.
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