Abstract
In an approach directed to isolate and characterize key proteins of the transduction cascade in photoreceptors using the phosphoinositide signaling pathway, we have isolated the Calliphora homolog of the Drosophila InaD gene product, which in Drosophila InaD mutants causes slow deactivation of the light response. By screening a retinal cDNA library with antibodies directed against photoreceptor membrane proteins, we have isolated a cDNA coding for an amino acid sequence of 665 residues (Mr = 73,349). The sequence displays 65.3% identity (77.3% similarity) with the Drosophila InaD gene product. Probing Western blots with monospecific antibodies directed against peptides comprising amino acids 272-542 (anti-InaD-(272-542)) or amino acids 643-655 (anti-InaD-(643-655)) of the InaD gene product revealed that the Calliphora InaD protein is specifically associated with the signal-transducing rhabdomeral photoreceptor membrane from which it can be extracted by high salt buffer containing 1.5 M NaCl. As five out of eight consensus sequences for protein kinase C phosphorylation reside within stretches of 10-16 amino acids that are identical in the Drosophila and Calliphora InaD protein, the InaD gene product is likely to be a target of protein kinase C. Phosphorylation studies with isolated rhabdomeral photoreceptor membranes followed by InaD immunoprecipitation revealed that the InaD protein is a phosphoprotein. In vitro phosphorylation is, at least to some extent, Ca 2+ dependent and activated by phorbol 12-myristate 13-acetate. The inaC-encoded eye-specific form of a protein kinase C (eye-PKC) is co-precipitated by antibodies specific for the InaD protein from detergent extracts of rhabdomeral photoreceptor membranes, suggesting that the InaD protein and eye-PKC are interacting in these membranes. Co-precipitating with the InaD protein and eye-PKC are two other key components of the transduction pathway, namely the trp protein, which is proposed to form a Ca2+ channel, and the norpA-encoded phospholipase C, the primary target enzyme of the transduction pathway. It is proposed that the rise of the intracellular Ca2+ concentration upon visual excitation initiates the phosphorylation of the InaD protein by eye-PKC and thereby modulates its function in the control of the light response.
Highlights
Phototransduction by rhabdomeral photoreceptors, of Drosophila compound eyes, has become an important model system for the ubiquitous phosphoinositide-mediated signal transduction
Isolation and Characterization of Calliphora InaD cDNAs—Antibodies directed against proteins of the fly photoreceptor membrane had been generated by immunizing rabbits with rhabdomes isolated from 2800 Calliphora eyes
We obtained 14 cDNA clones coding for the Calliphora homolog of InaD and determined the nucleotide sequence of the longest cDNA for both strands
Summary
Fly Stocks—Calliphora erythrocephala Meig., chalky mutant, was reared on bovine liver to maintain a high rhodopsin content in the eyes. Polyclonal anti-InaD antibodies were generated as follows: a DNA fragment encoding the 23 C-terminal amino acids of the Calliphora InaD protein was amplified by polymerase chain reaction from cloned cDNA using sequence-specific primers. Immunoprecipitation of the InaD Protein—Proteins were extracted from purified rhabdomeral membranes of 30 Calliphora eyes in 30 l of Triton X-100 buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris/HCl, pH 8.0, and 1 mM phenylmethylsulfonyl fluoride) for 15 min at 4 °C. Protein Phosphorylation and Dephosphorylation—The standard assay for protein phosphorylation was carried out in a buffer containing Hepes-buffered saline (115 mM NaCl, 2 mM KCl, 10 mM Hepes), pH 6.8, 2 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 125 M EGTA, 250 M CaCl2, and purified photoreceptor membranes from 10 fly retinae per sample. The amount of InaD protein present in the InaD protein band was calculated by laser densitometry using bovine serum albumin as a standard
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