The relative levels of GST and GST fusion proteins that bound and eluted from your beads (shown at the bottom) were determined by fractionating an equal volume of each eluate by SDS-PAGE and staining with Coomassie blue

The relative levels of GST and GST fusion proteins that bound and eluted from your beads (shown at the bottom) were determined by fractionating an equal volume of each eluate by SDS-PAGE and staining with Coomassie blue. However, elimination of any other member of the signalplex experienced no impact on the spatial distribution of INAD. A direct conversation between TRP and INAD did not appear to have a role in the photoresponse impartial of localization of multiple signaling components. Rather, the primary function of the TRP/ INAD complex is to form the core unit required for localization of the signalplex to the rhabdomeres. phototransduction (for reviews observe Pak 1995; Montell 1999). phototransduction is among the fastest of G proteinCcoupled cascades and is activated and terminated in tens of milliseconds (Ranganathan et al. 1991). The components that function in the light response are situated in the photoreceptor cells within a specialized 6-Thioinosine microvillar-containing organelle referred to as the rhabdomere. Many of the proteins that are critical for phototransduction are linked through conversation with the modular adaptor protein inactivation no afterpotential D (INAD; Huber et al. 1996a; Shieh and Zhu 1996; Chevesich et al. 1997; Tsunoda et al. 1997; Xu et al. 1998; Wes et al. 1999). INAD consists primarily of five 6-Thioinosine 90Camino acid protein conversation motifs called PDZ domains (Shieh and Niemeyer 1995). PDZ motifs occur in a large variety of proteins and bind to a diversity of signaling, cell adhesion, and cytoskeletal proteins (for reviews observe Kim 1997; Sheng and Wyszynski 1997; Craven and Bredt 1998; Dimitratos et al. 1999; Fanning and Anderson 1999; Schillace and Scott 1999). As a consequence of these interactions, PDZ-containing proteins nucleate macromolecular assemblies, and do so at specialized membrane structures such as synapses. In most cases, binding to PDZ domains is usually mediated through an S/T-X-V motif or hydrophobic or aromatic residues at the COOH termini of the binding partners (Kim et al. 1995; Kornau et al. 1995; Songyang et al. 1997; Daniels et al. 1998). A minimum of seven proteins binds directly to INAD. These include rhodopsin (Chevesich et al. 1997; Xu et al. 1998), phospholipase C (PLC; Huber et al. 1996a; Chevesich et al. 1997), protein kinase C (PKC; Huber et al. 1996a; Xu et al. 1998), the NINAC myosin III (Wes et al. 1999), calmodulin (Chevesich et al. 1997; Xu et al. 1998), and two light-dependent cation channel subunits, transient receptor potential (TRP; Shieh and Zhu 1996) and TRPL (Xu et al. 1998). Three of the binding proteins, TRP, PLC, and PKC, require conversation with INAD for localization in the rhabdomeres (Chevesich et al. 1997; Tsunoda et al. 1997). Mutation of the INAD binding sites in PLC (Shieh et al. 1997; van Huizen et al. 1998; Cook et al. 2000) CD47 and PKC (Adamski et al. 1998) causes defects in the photoresponse; however, the effects may be a consequence of instability and/or mislocalization of the proteins rather than 6-Thioinosine a specific requirement for tethering these proteins to INAD for proper signaling. Nevertheless, the signalplex also appears to have a direct role in signaling, independent of protein localization, as disruption of the NINAC/INAD conversation causes a delay in termination without causing any concomitant alteration in the concentration or the spatial distribution of NINAC (Wes et al. 1999). Despite considerable biochemical and genetic analyses of the signalplex, the determinants required in vivo for localization of INAD are not known. Candidate anchoring proteins include the light receptor (rhodopsin), ion channels (TRP and TRPL), and the cytoskeletal protein, NINAC. Here, we show that TRP and INAD share a reciprocal requirement for retention in the rhabdomeres. INAD was mislocalized in an age-dependent manner in strong alleles, or as a consequence of deleting the INAD 6-Thioinosine binding site in TRP. In contrast to a previous statement (Shieh and Zhu 1996), our results indicated that this direct conversation between TRP and INAD was mediated by the COOH-terminal residues. Furthermore, the TRP/INAD conversation was also required for the spatial distribution of two additional proteins, PLC and PKC, that depend.