Identifying IQGAP1 Domains Essential for Leukocyte Transmigration

Inflammation results in the dilation of local blood vessels, the activation of endothelium by cytokines and the recruitment of circulating leukocytes into areas of tissue damage. The majority of leukocytes undergo transendothelial migration (TEM) by using a unique endothelial cell membrane system referred to as the lateral border recycling compartment (LBRC). IQ-domain GTPase-activating protein 1 (IQGAP1) was identified as an LBRC-associated protein and subsequently found to be essential for TEM. IQGAP1 is a scaffolding protein comprised of six major interaction domains. It has been reported to interact productively with over 135 cellular proteins. In endothelial cells IQGAP1 partially localizes to cell borders and knocking down its expression prevents TEM. However, it is not known which of the many proteins it is known to interact with carries out its role in TEM. Since these proteins bind to specific IQGAP1 domains, as a first step toward understanding the mechanism by which IQGAP1 regulates TEM, we explored which of its six protein interactions domains were critical for its function. We knocked down IQGAP1 in endothelial cells and re-expressed truncated forms of IQGAP1 with specific regions deleted to identify the regions (and thereby the potential binding partners) that are critical for TEM. We show that the actin-binding domain and the calmodulin-binding isoleucine-glutamine (IQ) domain of IQGAP1 are critical for its function during TEM. Human endothelial cells in which >80% of endogenous IQGAP1 was depleted by shRNA did not support TEM; re-expression of wild-type IQGAP1 restored TEM to control levels. Re-expression of IQGAP1 with a GFP-tagged construct after knockdown also showed IQGAP1 enrichment around transmigrating leukocytes. Re-expression of IQGAP1 truncation mutants lacking the N-terminal actin-binding domain did not localize to endothelial borders and did not support TEM. Re-expression of truncated IQGAP1 constructs containing the actin-binding domain and the IQ domain localized to cell borders and supported TEM. Constructs containing the actin-binding domain but lacking the IQ domain localized to the cell borders but did not restore TEM. After identifying the IQ domain as having a critical role in TEM, we generated specific point mutations in this domain within the full-length construct. In particular, the isoleucine (I)-glutamine (Q) motif after which the IQ domain is named has been identified in the literature as having an essential role in IQGAP1 function and we are currently testing whether they are also important in TEM. Using these constructs in future studies will help identify the role of the IQ domain in TEM and its interacting partner(s). Identifying the binding partners of IQGAP1 that are critical for inflammation will provide clues to its mechanism of action and potential therapeutic targets for anti-inflammatory therapy.