Signaling Complexes of the FERM Domain-containing Protein GRSP1 Bound to ARF Exchange Factor GRP1

Abstract

GRP1 is a member of a family of proteins that contain a coiled-coil region, a Sec7 homology domain with guanosine nucleotide exchange activity for the ARF GTP-binding proteins, and a pleckstrin homology domain at the C terminus. The pleckstrin homology domain of GRP1 binds phosphatidylinositol (3,4,5) trisphosphate and mediates the translocation of GRP1 to the plasma membrane upon agonist stimulation of PI 3-kinase activity. Using a 32 P-labeled GRP1 probe to screen a mouse brain cDNA expression library, we isolated a cDNA clone encoding a GRP1-binding partner (GRSP1) that exists as two different splice variants in brain and lung. The GRSP1 protein contains a FERM protein interaction domain as well as two coiled coil domains and may therefore function as a scaffolding protein. Mapping experiments revealed that the interaction of GRP1 and GRSP1 occurs through the coiled coil domains in the two proteins. Immunodepletion experiments indicate that virtually all of the endogenous GRSP1 protein exists as a complex with GRP1 in lung. When co-expressed in Chinese hamster ovary cells expressing the human insulin receptor, both proteins display a diffuse, cytoplasmic localization. Acute translocation and co-localization of GRSP1 and GRP1 to ruffles in the plasma membrane was evident after insulin stimulation. These results identify GRSP1 as a novel member of GRP1 signaling complexes that are acutely recruited to plasma membrane ruffles in response to insulin receptor signaling.Numerous agonists acutely stimulate PI-3 kinase activities that generate the secondary messengers phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) 1 (1-6). The polyphosphoinositides control such diverse phenomena as membrane trafficking, chemotaxis, membrane ruffling, cell adhesion, prevention of apoptosis, differentiation, and oncogenic transformation of cells. To define downstream targets for these messengers, we isolated GRP1 by screening for clones that code for proteins that bind labeled 3Ј-phosphoinositides with high affinity (7). GRP1 contains a C-terminal PH domain that binds PtdIns(3,4,5)P 3 with high specificity and a central sec7 guanosine nucleotide exchange domain (8). A likely function of the molecule is to couple the production of PtdIns(3,4,5)P 3 in the cell membrane to activation of the small GTP-binding protein ARF6 (9 -11). GRP1 is part of a large family of proteins that include ARNO and cytohesin-1, which have been implicated in the regulation of integrin function (12), synaptic vesicle release (13), exocytosis in chromaffin cells (14), and desensitization of the luteinizing hormone receptor (15).The GRP1 family of proteins is recruited to the plasma membrane in many systems upon agonist stimulation by a mechanism that requires the presence of a functional PH domain and that is sensitive to inhibitors of PI-3 kinase (10,16,17). The initial interpretation was that recruitment occurs by direct binding to acutely generated PtdIns(3,4,5)P 3 . However, the mechani...

FRMD4A regulates epithelial polarity by connecting Arf6 activation with the PAR complex

“…GRSP-1 has been reported to bind to Grp-1 and colocalize with F-actin in membrane ruffles upon stimulation with insulin in CHO-T cells (22). The GFP-tagged GRSP-1 localized together with Par-3 at spot-like AJs and TJs in EpH4 cells and at AJs in NIH 3T3 cells (Fig.…”

The CNK1 scaffold binds cytohesins and promotes insulin pathway signaling

“…In addition to IPCEF1, the cytohesins have been found to interact with various other scaffold proteins, including GRASP/tamalin (Nevrivy et al 2000;Kitano et al 2002), CASP/Cybr/CYTIP (Mansour et al 2002;Tang et al 2002), and GRSP1 (Klarlund et al 2001). Although there is no primary sequence in common among these scaffolds, binding is mediated by the N-terminal coiled-coil (CC) domain present in the cytohesins and regions in each scaffold that also possess CC structure.…”

Differential Effects of Cytohesins 2 and 3 on β1 Integrin Recycling

“…IPCEF interacts with both ARNO and GRP1 by two-hybrid assay, although only the interaction with ARNO was confirmed with recombinant proteins (37). GRSP1 interacts with GRP1, and it is not known whether this protein can also bind to ARNO (38). It has been shown that these proteins co-localize with cytohesins and modify their ARF6-activating activity (37).…”