PHLPS AND PHLOPS IN THE PHOSDUCIN FAMILY OF G-BETA-GAMMA BINDING-PROTEINS

In this study, we identify new isoforms of the retinal phosducin and i nvestigate the expression of the phosducin family, showing that an iso form, PhLP1, has sequence homology with Phd and G beta gamma binding c apability, whereas two isoforms (phosducin-like orphan proteins, PhLOP s) share sequence homology with Phd but fail to bind G beta gamma. Ori ginal identification of PhLP1 and the PhLOPs was from a human retina c DNA library, using a PCR product for library hybridization screening t hat contained a predicted functional epitope domain. The screen identi fied Phd and three related, but distinct, recombinants (PhLP1, PhLOP1, and PhLOP2). By RT-PCR, all isoforms are expressed in either retina o r forskolin-stimulated Y79 retinoblastoma cells; however, the new isof orms are below the level of detection on Northern blot analysis. The p redicted amino acid translation of each homologue revealed major diffe rences, arising from either splice variants or gene duplication of Phd . To test the functional interaction of all phosducin isoforms with G beta gamma in vitro, a glutathione S-transferase (GST) fusion protein was developed for each member. Biochemical interaction with purified r etinal transducin G beta gamma was verified for GST-Phd and demonstrat ed for GST-PhLP1; however, neither GST-PhLOP1 nor GST-PhLOP2 bound G b eta gamma. Comparable results were observed when the GST-phosducin fus ion proteins selectively sequestered G beta gamma s from retinal extra cts or when functional G beta gamma interactions were assessed using s urface plasmon resonance technology. Phosducin and its isoforms are wi dely distributed in body tissues where they may participate in signal transduction pathways. Phd and PhLP1 possess an Il-amino acid conserve d epitope domain (TGPKGVINDWR) that controls the high-affinity binding of G beta gamma; these isoforms are implicated in the G-protein signa ling pathway. The phosducin-like orphan proteins (PhLOPs) fail to bind G beta gamma, suggesting that the PhLOP isoforms may participate in s till unidentified signaling pathways.