PhLPs and PhLOPs in the Phosducin Family of Gβγ Binding Proteins

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 Gβγ binding capability, whereas two isoforms (phosducin-like orphan proteins, PhLOPs) share sequence homology with Phd but fail to bind Gβγ. 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 Gβγ in vitro, a glutathione S-transferase (GST) fusion protein was developed for each member. Biochemical interaction with purified retinal transducin Gβγ was verified for GST−Phd and demonstrated for GST−PhLP1; however, neither GST−PhLOP1 nor GST−PhLOP2 bound Gβγ. Comparable results were observed when the GST−phosducin fusion proteins selectively sequestered Gβγs from retinal extracts or when functional Gβγ 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 Gβγ; these isoforms are implicated in the G-protein signaling pathway. The phosducin-like orphan proteins (PhLOPs) fail to bind Gβγ, suggesting that the PhLOP isoforms may participate in still unidentified signaling pathways