PH DOMAINS: DIVERSE SEQUENCES WITH A COMMON MEMBRANE ASSOCIATION FUNCTION. Kathryn M. Ferguson^#, Mark A. Lemmon⁺, Joseph Schlessinger^+, Paul B. Sigler^#, ^#Dept. of Molecular Biophysics and Biochemistry and the Howard Hughes Medical Institute, Yale University; ⁺Dept. of Pharmacology, New York University Medical Center

In efforts to understand intermolecular interactions that control signal transduction, we have studied the structure and function of pleckstrin homology (PH) domains. These domains of ~120 aa are found in some 90 different signal transduction proteins. Initially we studied the dynamin PH domain (DynPH). DynPH is a [[beta]]-sandwich of two orthogonal [[beta]]-sheets closed at one corner by a C-terminal [[alpha]]-helix. The X-ray crystal structure of DynPH confirmed that regions of sequence homology coincide with secondary structure elements, and that conserved hydrophobic residues, including the highly conserved W, form a well packed core. DynPH is electrostatically polarized, with three particularly variable loops coinciding with the positive face. These characteristics, common to all PH domains of known structure, suggest that PH domain ligands may be negatively charged, consistent with the previous suggestion that PH domains interact with anionic membrane components. The N-terminal region of phospholipase C-d₁, (PLCd₁) which contains a PH domain, has been implicated in binding to phosphatidylinositol-(4,5)-bisphosphate (PtdIns(4,5)P₂). We have shown that the isolated PH domain binds with high affinity and specificity to inositol-(1,4,5)-trisphosphate (Ins(1,4,5)P₃), and to PtdIns(4,5)P₂ in lipid vesicles. The X-ray crystal structure of PLCdPH complexed with Ins(1,4,5)P₃ shows that the ligand binds to the positive face, interacting with the three variable loops. No high affinity ligands have been identified for other PH domains. Recently, the phosphotyrosine binding (PTB) domain of the adapter protein Shc was shown to have a PH domain fold. The NPXpY binding motif, recognized by the Shc PTB domain, therefore represents a second high affinity PH domain ligand. Like PtdIns(4,5)P₂ binding to PLCd-PH, this interaction also targets the host protein (Shc) to the cell membrane.