Chemokines are critical for directing the traffic and organization of immune cells. We and others have shown that T cells migrate within lymphoid organs at high speeds. Under certain conditions, when migrating T cells encounter foreign antigen they form long-lived static conjugates with the antigen-presenting cell (APC), while at other times T cells detect foreign antigen but continue to migrate nonetheless. Understanding the determinants of these distinct phenotypes and their immunological consequences is critical for understanding the basic principles of the in vivo immune response. We have previously shown that some chemokines deliver dominant signals that directed T cells to continue migrating past antigen-bearing APCs, while other chemokines deliver subordinant signals that permit long-term conjugate formation. 2 such chemokines are SLC (CCL21, dominant) and SDF-1a (CXCL12, subordinate), recognized by their respective receptors on T cells, CCR7 and CXCR4. Here we propose to design chimeric receptors bearing the ligand-specificity 1 of a dominant receptor and the intracellular signaling response of a subordinate receptor. Because chemokine receptors are 7-transmembrane G-protein coupled receptors, their extracellular, transmembrane and intracellular structures are tightly coupled. In order to discover chimeras with functional ligand binding and appropriate intracellular signaling properties, we propose to generate a combinatorial library of receptor chimeras where all extracellular loops belong to 1 receptor, all intracellular loops belong to the other, and the transmembrane domains are varied between the 2.
In Aim 1 we will design and produce the transmembrane-shuffled CCR7/CXCR4 and CCR7/CCR5 libraries by a gene-synthesis approach.
In Aims 2 & 3 we will screen this library for desired chemotactic dominant/subordinate behavior.
In Aim 4 we will use this data to derive general principles for chemokine receptor transmembrane signaling and test these principles by rational design of a novel chimera between CXCR3 and CCR5. By elucidating the hierarchies of immunological """"""""Stop"""""""" and """"""""Go"""""""" signals we will reveal basic principles governing immune function that will be relevant to understand how tumors, viruses, and other microbes can sometimes evade immune clearance, and how immunity becomes dysregulated in autoimmune diseases. ? ? ?
| Kim, Natalie; Stiegler, Amy L; Cameron, Thomas O et al. (2008) Lrp4 is a receptor for Agrin and forms a complex with MuSK. Cell 135:334-42 |
