title: PhLPs and PhLOPs in the phosducin family of G beta gamma binding proteins.
abstract: In this study, we identify new isoforms of the retinal phosducin and investigate the expression of the phosducin family, showing that an isoform, PhLP1, has sequence homology with Phd and Gbeta gamma binding capability, whereas two isoforms (phosducin-like orphan proteins, PhLOPs) share sequence homology with Phd but fail to bind Gbeta gamma. Original identification of PhLP1 and the PhLOPs was from a human retina cDNA library, using a PCR product for library hybridization screening that contained a predicted functional epitope domain. The screen identified Phd and three related, but distinct, recombinants (PhLP1, PhLOP1, and PhLOP2). By RT-PCR, all isoforms are expressed in either retina or forskolin-stimulated Y79 retinoblastoma cells; however, the new isoforms are below the level of detection on Northern blot analysis. The predicted amino acid translation of each homologue revealed major differences, arising from either splice variants or gene duplication of Phd. To test the functional interaction of all phosducin isoforms with Gbeta gamma in vitro, a glutathione S-transferase (GST) fusion protein was developed for each member. Biochemical interaction with purified retinal transducin Gbeta gamma was verified for GST-Phd and demonstrated for GST-PhLP1; however, neither GST-PhLOP1 nor GST-PhLOP2 bound Gbeta gamma. Comparable results were observed when the GST-phosducin fusion proteins selectively sequestered Gbeta gammas from retinal extracts or when functional Gbeta gamma interactions were assessed using surface plasmon resonance technology. Phosducin and its isoforms are widely distributed in body tissues where they may participate in signal transduction pathways. Phd and PhLP1 possess an 11-amino acid conserved epitope domain (TGPKGVINDWR) that controls the high-affinity binding of Gbeta gamma; these isoforms are implicated in the G-protein signaling pathway. The phosducin-like orphan proteins (PhLOPs) fail to bind Gbeta gamma, suggesting that the PhLOP isoforms may participate in still unidentified signaling pathways.
authors.affiliation: Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, University of Southern California School of Medicine, Los Angeles 90033, USA. ccraft@hsc.usc.edu
authors.forename: C M
authors.initials: CM
authors.lastname: Craft
authors.forename: J
authors.initials: J
authors.lastname: Xu
authors.forename: V Z
authors.initials: VZ
authors.lastname: Slepak
authors.forename: X
authors.initials: X
authors.lastname: Zhan-Poe
authors.forename: X
authors.initials: X
authors.lastname: Zhu
authors.forename: B
authors.initials: B
authors.lastname: Brown
authors.forename: R N
authors.initials: RN
authors.lastname: Lolley
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020555:GTP-Binding Protein Regulators; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000096:biosynthesis; D012160:Retina; D012175:Retinoblastoma; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D009419:Nerve Tissue Proteins; C098789:PDCL protein, human; D010750:Phosphoproteins; D020033:Protein Isoforms; D012333:RNA, Messenger; C065631:phosducin; C113752:phosducin-like orphan protein-1; C113751:phosducin-like orphan protein-2; D019204:GTP-Binding Proteins