| Phototransduction in Drosophila melanogaster utilizes a G protein-coupled phospholipase C-mediated signaling pathway. The Drosophila no-r&barbelow;eceptor-p&barbelow;otential A&barbelow; (norpA) gene encodes a phosphatidylinositol-specific phospholipase C (PLC) enzyme that is essential for phototransduction. The NORPA protein is similar in overall structure and amino acid sequence to enzymes in the mammalian PLC-β family. NORPA protein is known to be activated by the α-subunit of a heterotrimeric G-protein (Gα).; Previous studies have shown that the central components of the Drosophila phototransduction pathway including the NORPA protein, protein kinase C (PKC), TRP protein (transient receptor potential; encoding an ion channel), and the TRPL protein (t&barbelow;ransient r&barbelow;eceptor p&barbelow;otential-l&barbelow;ike; encoding an ion channel), are organized via protein-protein interactions into a macromolecular signaling complex mediated by direct interactions with a multi-molecular scaffolding protein (called INAD; i&barbelow;nactivation-n&barbelow;o-a&barbelow;fterpotential D&barbelow;). G-protein appears to be indirectly integrated into this signaling complex through protein-protein interactions with the NORPA protein. The site for G-protein interaction with NORPA appears to reside within a segment of amino acids called the G-box, which is similar to the site that is required for G-protein activation of mammalian PLC. The putative G-box domain of NORPA is co-linear with a binding site that is essential for NORPA binding to INAD protein. Indeed, deletion of the G-box from the NORPA protein abolishes its ability to function in phototransduction, but does not abolish its basal PLC enzymatic activity. Thus, both G-protein and INAD appear to bind to NORPA protein at sites within the G-box. However, it is unknown if the binding sites for G-protein and WAD within the G-box of NORPA protein are adjacent, overlapping, or identical.; The purpose of the present study is to elucidate the sites of protein-protein interactions participating in formation of the signaling complex with a particular focus on elucidating sites of protein-protein interactions between NORPA, G-protein, and INAD. To accomplish this purpose, we carried out experimental work in the yeast two-hybrid system, a convenient model for studying protein-protein interactions. As a part of this study, we generated a series of deletions within the putative G-box domain of NORPA protein and expressed the altered proteins as Gal4 fusion proteins in yeast. Successful expression of the deletion constructs of NORPA protein in yeast were confirmed using immunoblotting assays. The Gα subunit of Drosophila phototransduction-specific G-protein and the INAD protein were also expressed as Gal4 fusion proteins in yeast. Results from the yeast two-hybrid assays show that deletion of segments of amino acids from #1013 to #1070 of NORPA protein eliminate binding to INAD protein. Conversely, deletion of segments of amino acids of NORPA protein from #993 to #1052 eliminate binding to the Gα subunit. Thus, the binding sites of NORPA to INAD and Gα appear to overlap, but may be adjacent. Yeast two-hybrid analyses show no direct interaction between INAD and Gα, which suggests that NORPA protein mediates an indirect interaction between INAD and Gα in the signaling complex. |
