This competitive renewal requests continued support for our investigation on the mechanisms of integrins - a class of heterodimeric (a/) transmembrane receptors. By binding to extracellular matrix (ECM) proteins via their extracellular domains and by connecting to cytoskeleton via their cytoplasmic tails (CTs), integrins are widely known to be the master regulators of cell adhesion and a variety of adhesion-dependent physiological and pathological responses. When expressed on the cell surface, integrin adopts latent state. Agonist stimulation, typically through a G-protein coupled receptor (GPCR), triggers specific intracellular signal(s) that are transmitted to the integrin cytoplasmic face, inducing an inside-out conformational activation of the receptor and its subsequent high affinity binding to ECM ligand. Upon ECM engagement, integrin is thought to transduce outside-in signal(s) back to the cytoplasm, triggering the reorganization of cytoskeleton. Such bi- directional signaling process not only supports firm cell adhesion but also allows the regulation of many dynamic adhesion processes such as cell spreading, migration, and proliferation. For several decades, intensive effort has been made to elucidate the molecular basis of this bi-directional signaling process, as reflected by hundreds of thousands of articles in PubMed. Our laboratory became interested in this topic in early 2000. Using NMR and cell biology approaches, we were the first to show that an integrin activator talin can disrupt a key integrin a/ cytoplasmic clasp, leading to the inside-out activation of the receptor. We further found recently that filamin, a major actin cross-linker, acts as an integrin inactivator by stabilizing the cytoplasmic clasp of inactive integrin. These findings have significantly advanced our understanding on the regulation of integrin, highlighting a balancing act between activator vs inactivator for dynamically controlling integrin activation and cell adhesion. However, two fundamental issues still remain unresolved: (1) upon the agonist stimulation, how are the signal(s) transmitted to integrin CTs to induce the receptor inside-out activation? (2) Upon activation and binding to ECM ligand, how does integrin transmit signal(s) back to the cytoplasm (outside-in signaling) to regulate the dynamics of cell adhesion? In preliminary studies, we discovered a large subgroup of GPCRs contain conserved filamin binding motif. Agonist-induced binding of select GPCR to filamin triggered integrin activation accompanied by filamin phosphorylation. This finding provides one exciting clue for Question 1, suggesting a novel filamin-mediated cross-talk between GPCR and integrin for regulating integrin inside-out signaling. Interestingly, we further found that in the activated state of integrin, the a CT adopts an altered conformation to re-associate with filamin, suggesting a novel structural basis of filamin-mediated integrin outside-in signaling, which may shed light upon the answer to Question 2. Thus, filamin appears to play a multi-faceted role in controlling the integrin bi-directional signaling.
In Aim1, we will further investigate the mechanism of GPCR-filamin interaction in regulating integrin activation. We will also elucidate the mechanism of GPCR-induced filamin phosphorylation and how it fine-tunes integrin activation (Aim2).
Aim3 will undertake detailed structural/functional analysis to examine how filamin mediates integrin outside-in signaling. We believe that the results derived from these aims will provide important clues for the long unanswered questions in integrin signaling. The studies may also help understand diseases associated with dysfunctions of integrin signaling and further promote development of therapeutics.
Integrin transmembrane receptor plays a central role in transmitting information between cell exterior and interior. Dysregulation of integrin signaling has been linked to several major human disorders including stroke, inflammation, and cancer. Our proposal will utilize state-of-the-art structural biology techniques coupled with cell biology assays to investigate long unresolved puzzles in integrin signaling by focusing a central integrin regulator filamin. Our studies will significantly improve our understanding on a wide range of integrin-mediated cellular responses such as cell migration and cell shape modulation. The studies will also impact on developing new strategies for treating integrin-associated diseases.
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