Lipid second messengers generated by phosphoinositide (PI) 3-kinases regulate diverse cellular functions through interaction with pleckstrin homology (PH) domains in modular signaling proteins. The PH domain of Grp1, a PI 3-kinase-activated exchange factor for Arf GTPases, selectively binds phosphatidylinositol 3,4,5-trisphosphate with high affinity. We have determined the structure of the Grp1 PH domain in the unliganded form and bound to inositol 1,3,4,5-tetraphosphate. A novel mode of phosphoinositide recognition involving a 20-residue insertion within the beta6/beta7 loop explains the unusually high specificity of the Grp1 PH domain and the promiscuous 3-phosphoinositide binding typical of several PH domains including that of protein kinase B. When compared to other PH domains, general determinants of 3-phosphoinositide recognition and specificity can be deduced.

For complex diseases in which multiple mediators contribute to overall disease pathogenesis by distinct or redundant mechanisms, simultaneous blockade of multiple targets may yield better therapeutic efficacy than inhibition of a single target. However, developing two separate monoclonal antibodies for clinical use as combination therapy is impractical, owing to regulatory hurdles and cost. Multi-specific, antibody-based molecules have been investigated; however, their therapeutic use has been hampered by poor pharmacokinetics, stability and manufacturing feasibility. Here, we describe a generally applicable model of a dual-specific, tetravalent immunoglobulin G (IgG)-like molecule--termed dual-variable-domain immunoglobulin (DVD-Ig)--that can be engineered from any two monoclonal antibodies while preserving activities of the parental antibodies. This molecule can be efficiently produced from mammalian cells and exhibits good physicochemical and pharmacokinetic properties. Preclinical studies of a DVD-Ig protein in an animal disease model demonstrate its potential for therapeutic application in human diseases.

Here we identified two novel proteins denoted EH domain protein 2 (EHD2) and EHD2-binding protein 1 (EHBP1) that link clathrin-mediated endocytosis to the actin cytoskeleton. EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1. Disruption of EHD2 or EHBP1 function by small interfering RNA-mediated gene silencing inhibits endocytosis of transferrin into EEA1-positive endosomes as well as GLUT4 endocytosis into cultured adipocytes. EHD2 localizes with cortical actin filaments, whereas EHBP1 contains a putative actin-binding calponin homology domain. High expression of EHD2 or EHBP1 in intact cells mediates extensive actin reorganization. Thus EHD2 appears to connect endocytosis to the actin cytoskeleton through interactions of its N-terminal domain with membranes and its C-terminal EH domain with the novel EHBP1 protein.

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