Abstract
Syntrophins are scaffold proteins of the dystrophin glycoprotein complex (DGC), which target ion channels, receptors, and signaling proteins to specialized subcellular domains. A yeast two-hybrid screen of a human brain cDNA library with the PSD-95, Discs-large, ZO-1 (PDZ) domain of gamma1-syntrophin yielded overlapping clones encoding the C terminus of TAPP1, a pleckstrin homology (PH) domain-containing adapter protein that interacts specifically with phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)). In biochemical assays, the C terminus of TAPP1 bound specifically to the PDZ domains of gamma1-, alpha1-, and beta2-syntrophin and was required for syntrophin binding and for the correct subcellular localization of TAPP1. TAPP1 is recruited to the plasma membrane of cells stimulated with platelet-derived growth factor (PDGF), a motogen that produces PI(3,4)P(2). Cell migration in response to PDGF stimulation is characterized by a rapid reorganization of the actin cytoskeleton, which gives rise to plasma membrane specializations including peripheral and dorsal circular ruffles. Both TAPP1 and syntrophins were localized to PDGF-induced circular membrane ruffles in NIH-3T3 cells. Ectopic expression of TAPP1 potently blocked PDGF-induced formation of dorsal circular ruffles, but did not affect peripheral ruffling. Interestingly, coexpression of alpha1- or gamma1-syntrophin with TAPP1 prevented the blockade of circular ruffling. In addition to syntrophins, several other proteins of the DGC were enriched in circular ruffles. Collectively, our results suggest syntrophins regulate the localization of TAPP1, which may be important for remodeling the actin cytoskeleton in response to growth factor stimulation.
Highlights
Many cellular signaling pathways involve dynamic changes in lipid composition at discrete sites in the plasma membrane [1]
Cell migration in response to platelet-derived growth factor (PDGF) stimulation is characterized by a rapid reorganization of the actin cytoskeleton, which gives rise to plasma membrane specializations including peripheral and dorsal circular ruffles
Cell migration in response to PDGF stimulation is characterized by a rapid reorganization of the actin cytoskeleton, which gives rise to several types of plasma membrane specializations including lamellipodia, peripheral membrane ruffles, and dorsal circular ruffles [21]
Summary
Materials—FuGENETM 6 transfection reagent was purchased from Roche Diagnostics. Dulbecco’s modified Eagle’s medium was purchased from Invitrogen Life Technologies, Inc. A polyclonal antibody to BAS-GRIP ( known as TAPP1; see below) was made by immunizing rabbits with a glutathione S-transferase (GST) fusion protein corresponding to the C-terminal 115 amino acids. The PDZ domain bait consisted of amino acids 51–149 of human ␥1-syntrophin fused in-frame with the LexA DNA binding domain in vector pHybLex/Zeo (Invitrogen). A full-length T7-tagged ␥1-syntrophin construct described previously [24] was digested with the same enzymes and ligated with the cut vector fragment. A cDNA clone was isolated in a yeast two-hybrid screen of a human kidney library with the second PDZ domain of PTP-BAS (amino acids 1206 –1496) as bait [25]. Preparation and Purification of Bacterial Fusion Proteins—Fusion proteins were purified as described previously [26] with the following modifications: BL21(DE3)pLysS cells were transformed and grown in. The number of cells with peripheral or dorsal circular ruffles was determined and expressed as the percentage of cells with ruffles
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