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Thrombin has a critical role in many adult and embryologic cellular processes, exerting its effects through two high-affinity thrombin receptor systems: protease-activated receptor 1 (PAR1) and the PAR3/PAR4 system. Both hPAR1 and hPAR3 are coclustered in the human genome, with hPAR3 encompassed within hIQGAP2, a putative GTPase activating protein with actin polymerizing functions linked to cytoskeletal reorganization. Since hPARs colocalize with hIQGAP2 in the human genome and function coordinately with this protein in platelet thrombin signaling pathways, we have further characterized these genes in developing embryonic and adult tissues. We confirmed the presence of a mIQGAP2/ mPAR gene cluster on murine Chromosome 13 and showed it to be organized similarly to that in humans, except that murine PAR3 is translated off the forward (sense) strand. Northern analysis demonstrated limited mPAR3 expression in adult tissues, although its expression during embryogenesis was evident at E15 in cartilage, brain, and keratinocytes. mIQGAPs 1 and 2 had congruent expression patterns in 11 of 15 adult tissues studied. In contrast, whole embryos demonstrated predominant mIQGAP1 expression starting at E7 and evident to E17. In situ hybridization of whole embryos (E9-E16) demonstrated distinct patterns of tissue-dependent mIQGAP1/ mIQGAP2 expression. Concordant expression (absence or presence) of mPAR1 with either mIQGAP1 or mIQGAP2 was seen in the majority (12 of 15) of adult tissues studied. Similarly, there was no evidence for mPAR3 expression during embryogenesis in the absence of either mIQGAP1 or mIQGAP2. These data provide a panoramic survey of PAR/ IQGAP expression as an initial approach to dissect thrombin signaling pathways linked to cytoskeletal reorganization.

Human blood platelets are anucleate cells whose response to extracellular stimuli results in actin cytoskeleton rearrangements, thereby providing the critical initial step in the regulation of hemostasis. The serine protease alpha-thrombin, known to activate platelets by cleavage of a family of protease-activated receptors (PARs), is the most potent physiologic activator of human platelets, though downstream effector proteins uniquely linked to platelet cytoskeletal actin polymerization remain largely uncharacterized. The gene encoding the putative rac1/cdc42 effector protein IQGAP2 was identified within the PAR gene cluster at 5q13, flanked telomeric by PAR1 and encompassing PAR3. Immunofluorescence microscopy demonstrated IQGAP2 expression in filopodial extensions of activated platelets and colocalized with F-actin in lamellipodia and filopodia of IQGAP2-transfected COS1 cells. Platelet activation by alpha-thrombin, but not saturating concentrations of fibrillar collagen or adenosine 5'-diphosphate, uniquely assemble an IQGAP2/arp2/3-actin cytoplasmic complex, an association regulated by guanosine triphosphate rac1 ([GTP]rac1) but not by [GTP]cdc42. Likewise, only thrombin-activated platelets resulted in rapid translocation of IQGAP2 to the platelet cytoskeleton. These observations identify a physiologic scaffolding function for IQGAP2 and establish the presence of a functional genomic unit in humans uniquely evolved to regulate thrombin-induced platelet cytoskeletal actin reorganization.

IQ motif-containing GTPase-activating protein 2 (IQGAP2) is a multidomain scaffolding protein that plays a role in cytoskeleton regulation by juxtaposing Rho GTPase and Ca2+/calmodulin signals. While IQGAP2 suppresses tumorigenesis in liver, its role in pathophysiology of the gastrointestinal tract remains unexplored. Here we report that IQGAP2 is required for the inflammatory response in colon. Mice lacking Iqgap2 gene (Iqgap2-/- mice) were resistant to chemically-induced colitis. Unlike wild-type controls, Iqgap2-/- mice treated with 3% dextran sulfate sodium (DSS) in water for 13 days displayed no injury to colonic epithelium. Mechanistically, resistance to colitis was associated with suppression of colonic NF-κB signaling and IL-6 synthesis, along with diminished neutrophil and macrophage production and recruitment in Iqgap2-/- mice. Finally, alterations in IQGAP2 expression were found in colons of patients with inflammatory bowel disease (IBD). Our findings indicate that IQGAP2 promotes inflammatory response at two distinct levels; locally, in colonic epithelium through TLR4/NF-κB signaling pathway, and systemically, via control of maturation and recruitment of myeloid immune cells. This work identifies a novel mechanism of colonic inflammation mediated by signal transducing scaffolding protein IQGAP2. IQGAP2 domain-specific blocking agents may represent a conceptually novel strategy for therapy of IBD and other inflammation-associated disorders, including cancer. [ABSTRACT FROM AUTHOR]