Anchoring TRP to the INAD macromolecular complex requires the last 14 residues in its carboxyl terminus.
Drosophila transient-receptor-potential (TRP) is a Ca2+ channel responsible for the light-dependent depolarization of photoreceptors. TRP is anchored to a macromolecular complex by tethering to inactivation-no-afterpotential D (INAD). We previously reported that INAD associated with the carboxyl tail of TRP via its third post-synaptic density protein 95, discs-large, zonular occludens-1 domain. In this paper, we further explored the molecular basis of the INAD interaction and demonstrated the requirement of the last 14 residues of TRP, with the critical contribution of Gly1262, Val1266, Trp1274, and Leu1275. We also revealed by pull-down assays that the last 14 residues of TRP comprised the minimal sequence that competes with the endogenous TRP from fly extracts, leading to the co-purification of a partial INAD complex containing INAD, no-receptor-potential A, and eye-protein kinase C (PKC). Eye-PKC is critical for the negative regulation of the visual signaling and was shown to phosphorylate TRP in vivo. To uncover the substrates of eye-PKC in the INAD complex, we designed a complex-dependent eye-PKC assay, which utilized endogenous INAD complexes isolated from flies. We demonstrate that activated eye-PKC phosphorylates INAD, TRP but not no-receptor-potential A. Moreover, phosphorylation of TRP is dependent on the presence of both eye-PKC and INAD. Together, these findings indicate that stable kinase-containing protein complexes may be isolated by pull-down assays, and used in this modified kinase assay to investigate phosphorylation of the proteins in the complex. We conclude that TRP associates with INAD via its last 14 residues to facilitate its regulation by eye-PKC that fine-tunes the visual signaling.
Peng
Li
L
Department of Pharmacology, Center for Molecular Neuroscience, and Vanderbilt Vision Research Center, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Popescu
Daniela C
DC
Wang
Ning
N
Shieh
Bih-Hwa
BH
D000818:Animals; D004330:Drosophila; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D046911:Macromolecular Substances / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D054799:Phospholipase C beta / Q000378:metabolism; D010766:Phosphorylation; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism; D050051:Transient Receptor Potential Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism; D014785:Vision, Ocular / Q000502:physiology*; D050994:Voltage-Dependent Anion Channels
D029721:Drosophila Proteins; D005136:Eye Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D050051:Transient Receptor Potential Channels; D050994:Voltage-Dependent Anion Channels; C091568:inaD protein, Drosophila; C063169:trp protein, Drosophila; D012694:Serine; D014364:Tryptophan; D011493:Protein Kinase C; C468371:NorpA protein, Drosophila; D054799:Phospholipase C beta; D007930:Leucine; D005998:Glycine