Elucidation of the Interaction of Calmodulin with the IQ Motifs of IQGAP1

Abstract

Calmodulin regulates the function of numerous proteins by binding to short regions on the target molecule. IQ motifs, which are found in over 100 human proteins, appear in tandem repeats and bind calmodulin in the absence of Ca 2؉ . One of these IQ-containing proteins, IQGAP1, interacts with several targets, including Cdc42, ␤-catenin, E-cadherin, and actin, in a calmodulin-regulated manner. To elucidate the molecular mechanism by which apocalmodulin and Ca 2؉ /calmodulin differentially regulate IQGAP1, a series of constructs of IQGAP1 with selected point mutations of the four tandem IQ motifs were generated. Mutating the basic charged arginine residues in all four IQ motifs abrogated binding of IQGAP1 to apocalmodulin, but had no effect on its interaction with Ca 2؉ /calmodulin. Analysis of IQGAP1 constructs with point mutations in single, double, or triple IQ motifs revealed that apocalmodulin bound only to IQ3 and IQ4. By contrast to the arginine mutant constructs, mutation of selected hydrophobic residues in the IQ motifs produced an IQGAP1 protein incapable of binding either apocalmodulin or Ca 2؉ /calmodulin. These results, which differ from the conventional model of Ca 2؉ -independent binding of calmodulin to IQ motifs, provide insight into the complexity of the molecular interactions between calmodulin and IQ motifs.

Multiple proteins mediate IQGAP1-stimulated cell migration

“…This hypothesis was confirmed by the analyses with the point mutant IQGAP1 constructs that selectively lack binding to calmodulin. Transfection of IQGAP1·apoCaM (−) , which cannot bind apocalmodulin but binds normally to Ca 2+ /calmodulin [21], increases cell migration by essentially the same magnitude as wild type IQGAP1. In contrast, transfection of IQGAP1·CaM (−) , a point mutant IQGAP1 construct that fails to interact with Ca 2+ /calmodulin [21], augments cell migration substantially more than wild type IQGAP1.…”

“…To eliminate all calmodulin binding, selected hydrophobic residues and the amino acid immediately proximal to the glutamine (the "I" of the IQ) were replaced in each IQ motif. These constructs have been described and characterized in detail previously [21]. Overexpression of IQGAP1·apoCaM (−) enhanced cell migration by essentially the same magnitude as that produced by wild type IQGAP1 (Fig.…”

“…The construction of IQGAP1·apoCaM (−) and IQGAP1·CaM (−) , point mutant constructs that lack binding to apocalmodulin and calmodulin, respectively, has been described previously [21]. IQGAP1G75Q was generated by site-directed mutagenesis using Strategene Quick Change site-directed mutagenesis method.…”

“…Therefore, we examined the effect of calmodulin on IQGAP1-stimulated cell migration. Analysis was performed with two point-mutant IQGAP1 constructs we developed: IQGAP1·CaM (−) , which is unable to bind calmodulin, and IQGAP1·apoCaM (−) , which lacks binding to apocalmodulin (Ca 2+ -free calmodulin) but binds Ca 2+ /calmodulin [21]. Replacement of basic charged arginine residues in each of the four IQ motifs (and glutamine in the second IQ motif) yields a construct to which apocalmodulin does not bind.…”

IQGAP1 Protein Regulates Nuclear Localization of β-Catenin via Importin-β5 Protein in Wnt Signaling

“…IQGAP1 is a 190-kDa and contains multiple protein-interacting domains; the CHD (calponin homology) domain binds to actin, the WW domain binds to ERK2, the IQ repeat motifs bind to calmodulin and myosin light chain, the RasGAP-like domain binds to Cdc42 and Rac1, and the RasGAP_c domain at the C terminus binds to ␤-catenin and E-cadherin (15)(16)(17)(18)(19)(20)(21). IQGAP1 is involved in various cellular processes including cytoskeletal reorganization, cell adhesion, and cell cycle (22).…”