Characterization of Human Alpha-Dystrobrevin Isoforms in HL-60 Human Promyelocytic Leukemia Cells Undergoing Granulocytic Differentiation

The biochemical properties and spatial localization of the protein alpha-dystrobrevin and other isoforms were investigated in cells of the human promyelocytic leukemia line HL-60 granulocytic differentiation as induced by retinoic acid (RA). Alpha-dystrobrevin was detected both in the cytosol and the nuclei of these cells, and a short isoform (gamma-dystrobrevin) was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of alpha-dystrobrevin and its isoforms could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and human neutrophils. Moreover, the gamma-dystrobrevin isoform was found in association with actin and myosin light chain. The results provide new information about potential involvement of alpha-dystrobrevin and its splice isoforms in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells

A Conformational Switch in the Scaffolding Protein NHERF1 Controls Autoinhibition and Complex Formation*

The mammalian Na+/H+ exchange regulatory factor 1 (NHERF1) is a multidomain scaffolding protein essential for regulating the intracellular trafficking and macromolecular assembly of transmembrane ion channels and receptors. NHERF1 consists of tandem PDZ-1, PDZ-2 domains that interact with the cytoplasmic domains of membrane proteins and a C-terminal (CT) domain that binds the membrane-cytoskeleton linker protein ezrin. NHERF1 is held in an autoinhibited state through intramolecular interactions between PDZ2 and the CT domain that also includes a C-terminal PDZ-binding motif (-SNL). We have determined the structures of the isolated and tandem PDZ2CT domains by high resolution NMR using small angle x-ray scattering as constraints. The PDZ2CT structure shows weak intramolecular interactions between the largely disordered CT domain and the PDZ ligand binding site. The structure reveals a novel helix-turn-helix subdomain that is allosterically coupled to the putative PDZ2 domain by a network of hydrophobic interactions. This helical subdomain increases both the stability and the binding affinity of the extended PDZ structure. Using NMR and small angle neutron scattering for joint structure refinement, we demonstrate the release of intramolecular domain-domain interactions in PDZ2CT upon binding to ezrin. Based on the structural information, we show that human disease-causing mutations in PDZ2, R153Q and E225K, have significantly reduced protein stability. Loss of NHERF1 expressed in cells could result in failure to assemble membrane complexes that are important for normal physiological functions