PHR1 Encodes an Abundant, Pleckstrin Homology Domain-containing Integral Membrane Protein in the Photoreceptor Outer Segments

Shunbin Xu,Rahim Ladak,Deborah A Swanson,Anna Soltyk,Hui Sun,Lynda Ploder,Danka Vidgen,Alessandra M.V Duncan,Elizabeth Garami,David Valle,Roderick R Mcinnes

doi:10.1074/jbc.274.50.35676

Abstract

We cloned human and murine cDNAs of a gene (designated PHR1), expressed preferentially in retina and brain. In both species, PHR1 utilizes two promoters and alternative splicing to produce four PHR1 transcripts, encoding isoforms of 243, 224, 208, and 189 amino acids, each with a pleckstrin homology domain at their N terminus and a transmembrane domain at their C terminus. Transcript 1 originates from a 5'-photoreceptor-specific promoter with at least three Crx elements ((C/T)TAATCC). Transcript 2 originates from the same promoter but lacks exon 7, which encodes 35 amino acids immediately C-terminal to the pleckstrin homology domain. Transcripts 3 and 4 originate from an internal promoter in intron 2 and either include or lack exon 7, respectively. In situ hybridization shows that PHR1 is highly expressed in photoreceptors, with lower expression in retinal ganglion cells. Immunohistochemistry localizes the PHR1 protein to photoreceptor outer segments where chemical extraction studies confirm it is an integral membrane protein. Using a series of PHR1 glutathione S-transferase fusion proteins to perform in vitro binding assays, we found PHR1 binds transducin betagamma subunits but not inositol phosphates. This activity and subcellular location suggests that PHR1 may function as a previously unrecognized modulator of the phototransduction pathway.

Highlights

In response to a single photon, mammalian photoreceptors produce an electrochemical signal that is integrated with many others, transmitted to higher visual centers in the brain, and perceived as vision
To confirm and extend our initial result, we examined the expression of PHR1 on Northern blots of RNA from multiple human tissues (Fig. 1A)
We found an ϳ2.0-kb PHR1 transcript(s) highly expressed in retina and brain, with much lower expression in several other tissues and no detectable expression in lymphoblasts or fibroblasts

Summary

EXPERIMENTAL PROCEDURES

Cloning, Sequencing, and 5Ј-RACE Extension on PHR1—We performed a differential hybridization screen on a human retinal cDNA library as described [21]. Following three washes in PBS, we prehybridized the acetylated sections by incubation in hybridization buffer (50% formamide, 5ϫ SSC, 5ϫ Denhardt’s solution (Sigma), 250 ␮g/ml MRE600 tRNA (Roche Molecular Biochemicals), 500 ␮g/ml herring sperm DNA (Promega) at room temperature for 6 h in a 5ϫ SSC humidified chamber. Fusion Proteins—To make fusion protein constructs, we amplified full-length or segments of human PHR1 cDNA and cloned the products in pMAL-C2 (New England Biolab) or pGST/His-T1 (Amersham Pharmacia Biotech). We incubated the sections in primary antibodies at 4 °C overnight, followed by washing with PBS for 10 min three times and incubation with fluorescein-conjugated secondary antibody at room temperature for 1 h. We electroblotted the proteins to Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech) with a semi-dry transfer cell (Bio-Rad) and probed the blots with anti-G␤ (Santa Cruz Biotechnology, Inc.) and horseradish peroxidase-conjugated goat-anti-rabbit IgG (Bio-Rad) as secondary antibody using ECL Western blotting detection system (Amersham Pharmacia Biotech). We suspended the final pellet in 400 ␮l of Solvable (Packard), added 10 ml of Aquassure (Packard) and glacial acetic acid (400 ␮l) to suppress chemiluminescence, and quantified the radioactivity by liquid scintillation spectrometry

RESULTS

Splicing acceptor

DISCUSSION