TI - CD317 interacts with RICH2 , a protein with both a Bin/amphiphysin/Rvs ( BAR ) and Rho GTPase-activating protein ( GAP ) domain . AB - Having shown that knockdown of CD317 expression leads to a change in organization of the actin cytoskeleton and a concomitant change in localization of ezrin and having implicated Rac and Rho activity in this phenotype , we reasoned that there might be some physical link between CD317 and ezrin and /or Rac and /or Rho or possibly with the actin cytoskeleton itself . Therefore , we chose to screen for proteins that interact with the cytosolic domain of CD317 . Our chosen strategy was to use a synthetic peptide , corresponding to the cytosolic domain of CD317 and tethered to a matrix support at its C terminus , to screen a bacteriophage display library ( see Materials and methods ) . The rationale for screening a bacteriophage display library rather than a yeast two hybrid library was that we reasoned that any interaction with the cytosolic domain of CD317 might require the free N terminus of CD317 ; this would be blocked by fusion to the yeast partner protein in any yeast two hybrid bait construct but would remain exposed in the C-terminally tethered peptide that was used to screen the bacteriophage display library . The screen identified 19 sequences encoding potential interactors . This included several proteins we considered to be improbable genuine interactors with CD317 ( eg , hypothetical proteins and ribosomal proteins ; Fig S2 D ) . However , one protein identified as a candidate interactor was RICH2 ( Richnau and Aspenstrom , 2001 ) . Several features of RICH2 ( see the next paragraph ) suggested that it might be a genuine interactor with the CD317 cytosolic domain . Therefore , we used three further techniques to confirm the CD317-RICH2 interaction . The first was a pull-down assay in which a synthetic peptide , biotinylated at its C terminus and corresponding to the entire cytosolic N terminus of CD317 , was immobilized on streptavidin-coated beads and used to isolate a fusion protein of the C-terminal 87 amino acids of RICH2 fused to GST ( Fig 4 A ) . Biotin-coated beads did not bind the GST-RICH2 fusion ( Fig 4 A ) , and GST did not bind to the beads coated with the CD317 peptide . The second technique was coimmunoprecipitation . In initial experiments , CD317-GFP was immunoprecipitated using an anti-GFP antibody from COS cells expressing CD317-GFP and RICH2-RFP , and the immunoprecipitated material was probed with an antibody to RICH2 ( Fig 4 B , top ) . In subsequent experiments , endogenous CD317 was immunoprecipitated from polarized Caco-2 cells ( or CD317 knockdown Caco-2 cells ) , and the immunoprecipitated material was probed with an antibody to RICH2 ( Fig 4 B , bottom ) . RICH2 was detected in material isolated from control cells but not from that isolated from the CD317 knockdown cells , demonstrating that endogenous RICH2 interacts with endogenous CD317 . The third technique used to confirm the CD317-RICH2 interaction was surface plasmon resonance . The biotinylated synthetic peptide corresponding to the cytosolic N terminus of CD317 was immobilized on a streptavidin-coated gold chip , and the GST-RICH2 fusion protein passed over the chip at different concentrations . The GST-RICH2 fusion protein can be seen to bind to the immobilized CD317 peptide in a dose-responsive manner ( Fig 4 C ) . Thus , all three techniques confirmed that RICH2 interacts with the cytosolic domain of CD317 and experiments using the GST-RICH2 fusion demonstrate that the C-terminal 87 amino acids of RICH2 are involved in that interaction . RICH2 was originally identified as a homologue of RICH1 ( also known as Nadrin and ARHGAP17 [Katoh and Katoh , 2004] ; Richnau and Aspenstrom , 2001 ) . RICH2 , different isoforms of RICH1 , and related proteins share two conserved domains , namely an N-terminal BAR domain and a Rho/Rac/cdc42 GAP domain . These domains are followed by a variable ( in sequence and length ) region ( Fig 4 D ) . However , RICH2 and isoforms 1 and 2 of RICH1 do share the same C-terminal sequence Glu-Ser-Thr-Ala-Leu (ESTAL) , which conforms to a PSD-95/DlgA/ZO-1-like ( PDZ ) domain -binding motif and has been implicated in binding to the protein EBP50 ( ERM -binding PHOSphoprotein 50 ; also known as NHERF1 [Na+/H+ exchanger regulatory factor 1] ; Reczek and Bretscher , 2001 ) . EBP50 is a PDZ domain -containing adapter that has been shown to link ERM proteins with the cytosolic domains of specific integral membrane proteins . In this case , RICH2 appears to provide a physical link between the cytosolic domain of CD317 and EBP50 and thus to ezrin and the actin cytoskeleton . We reasoned that if this were the case , it should be possible to coimmunoprecipitate EBP50 with CD317 . We immunoprecipitated CD317 from lysate from polarized Caco-2 cells ( control or CD317 knockdown ) and immunoblotted the immunoprecipitates using an anti-EBP50 antibody . EBP50 was detectable in the immunoprecipitate from control cells but not in that from CD317 knockdown cells ( Fig 4 E and Fig S2 C ) . RICH2 also appears to provide a potential link between CD317 and regulation of Rac and Rho because the central putative GAP domain has been shown to have Rac GAP activity in vitro ( Richnau and Aspenstrom , 2001 ) . The BAR domain of RICH2 has been shown to bind membranes and to be capable of inducing membrane tubulation . Consistent with this in vitro observation , we found RICH2 to be present in the membrane fraction from Caco-2 cells after the separation of membrane and cytosol fractions ( Fig 4 F ) . This was also the case for RICH2 in CD317 knockdown Caco-2 cells ( unpublished data ) ; thus , CD317 is not required for the membrane association of RICH2 .