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

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-photoreceptorspecific 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 ␤␥ subunits but not inositol phosphates. This activity and subcellular location suggests that PHR1 may function as a previously unrecognized modulator of the phototransduction pathway.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 ultimately perceived as vision. This elegant system depends on the coordinated expression of a large set of genes and on the interaction of their protein products. Many components of this system are expressed only in photoreceptors, and several are associated with retinal degeneration when defective. Mutations in genes encoding proteins involved in the phototransduction pathway (e.g. rhodopsin (Refs. 1 and 2), transducin (Refs. 3 and 4), or phosphodiesterase (Refs. 5-8)), photoreceptor structure (e.g. peripherin/RDS and ROM1 (Refs. 9 -12)), interactions between photoreceptors and RPE 1 (e.g. ABCR (Refs. 13-15)), or in photoreceptor-specific gene expression (e.g. CRX) (Refs. 16 -18)) result in inherited retinal degenerations (19,20). Accordingly, we reasoned that genes expressed preferentially or exclusively in retina would be important for the normal retinal structure and function, and would be likely candidates for retinal degenerations. To identify genes with these expression characteristics, we implemented a differential hybridization screen (9, 21). One of the clones identified in this screen, designated PHR1 (for pleckstrin homology domain retinal protein) was of interest because it showed preferential expression in retina and brain. We subsequently cloned and sequenced human and murine full-length PHR1 cDNAs and found that they encode proteins with an N-terminal PH domain and a hydrophobic, putative transmembrane domain at the C terminus. PHR1 is the first PH domain-containing protein found to be...

Phosphoinositide 3-kinase signaling in the vertebrate retina

“…Evictin-1 can localize to rhodopsin-laden, postGolgi membranes in photoreceptor cells, suggesting that the protein may be involved in postGolgi traffi cking of membranes ( 202 ). PHR1 is present in outer segments and binds to transducin ␤ ␥ subunits but does not bind to inositol phosphates; however, PI lipids were not tested ( 204 ). The PH domain of PHR1 is most similar to that of Akt, which we found in the ROS ( 135 ).…”

“…The best examples are Akt isoforms ( 52,135 ), IRS-1, IRS-2 ( 52, 68, 69 ), Grb14 ( 157 ), Evictin-1 ( 202 ), PHR1 ( p leckstrin h omology domain r etinal protein) ( 203,204 ), and recently identifi ed Akt dephosphorylating enzymes PHLPP (PH domain and leucine rich repeat protein phosphatases) and PHLPPL (PH domain and leucine rich repeat protein phosphatase-like) ( 205 ). Deletion of two PI-binding proteins, Akt2 ( 135 ) and IRS-2 ( 69 ), in the retina results in photoreceptor degeneration.…”

PHR1, an integral membrane protein of the inner ear sensory cells, directly interacts with myosin 1c and myosin VIIa

“…In the cochlea PHR1 is also expressed in a subset of interdental cells at the surface of the spiral limbus (Xu et al, 2004). Immunohistochemistry and chemical extraction studies on the photoreceptor outer segment have shown that PHR1 is an integral membrane protein (Xu et al, 1999). PHR1 contains an N-terminal pleckstrin homology domain (Krappa et al, 1999;Xu et al, 1999).…”

“…We identified 71 clones by histidine nutritional selection and β-galactosidase (β-gal) activity. The inserts from the isolated clones were sequenced, leading to the identification of four overlapping clones that encode the C-terminal region of a protein named PHR1 (Krappa et al, 1999;Xu et al, 1999) (Fig. 1B).…”

“…These include the mechanotransduction slow adaptation process that takes place in the stereocilia of mature hair cells . Here, we show that PHR1, an integral membrane protein expressed in the hair cells (Xu et al, 1999;Xu et al, 2004), directly interacts with the myosin 1c and myosin VIIa tails, and is able to dimerise. We discuss the functional implications of these findings in the inner ear sensory cells.…”