The roles of the phosducin family proteins in the regulation of heterotrimeric G proteins in vertebrate photoreceptors

Phosducin (Pdc) and phosducin-like protein 1 (PhLP1) are homologous proteins of the phosducin gene family that specifically interact with heterotrimeric G proteins. Both Pdc and PhLP1 are expressed in photoreceptors, sensory neurons of the retina responsible for acquisition of visual information; however their functions in these cells remains enigmatic. Photoreceptors maintain remarkably high and tightly controlled levels of the heterotrimeric G protein, transducin, utilized by these cells for visual signal transduction. Our central hypothesis was that Pdc and PhLP1 are engaged in the maintenance of transducin homeostasis in the photoreceptors.;To test this hypothesis, we have studied phosphorylation of Pdc, which regulates its interaction with transducin in a light-dependent manner. For that, we have determined light-dependence, time-course, and localization of phosphorylated Pdc within the photoreceptor cell under various physiologically relevant conditions of illumination. We found that phosphorylation of Pdc in vivo occurs in a site- and compartment-specific manner, and is specifically enriched in the border between the inner and outer segments of rod photoreceptors. These findings are described in Part I of Chapter 2.;An abundance of Pdc phosphorylation at the entrance to the outer segment allowed us to hypothesize that Pdc regulates trafficking of transducin to this compartment. To test our hypothesis directly, we generated transgenic mice expressing Pdc lacking the principal phosphorylation site, serine 54 and serine 71, under the control of a rhodopsin promoter. Using this Pdc phosphorylation mutant, and transgenic mice expressing full-length Pdc as a control, we compared the rates of transducin trafficking to the rod outer segments, and found that phosphorylation of Pdc significantly accelerates trafficking of transducin to the rod outer segments. This ongoing research project is described in Part II of Chapter 2.;To explore the role of PhLP1, we have suppressed its endogenous activity in photoreceptors using transgenic overexpression of a dominant-negative N-terminally truncated splice-isoform of PhLP1. We found that suppressing PhLP1 activity triggered fast and severe photoreceptor degeneration, apparently due to a profound disruption in transducin expression. These findings, described in Chapter 3, strongly support our hypothesis that PhLP1 plays a central role in the post-translational stabilization of transducin subunits, which is essential for visual function and rod viability.;In summary, our findings have demonstrated that phosducin and phosducin-like protein 1 function as specific chaperones in the folding, assembly and trafficking of transducin subunits in photoreceptors

Phosducin regulates the expression of transducin betagamma subunits in rod photoreceptors and does not contribute to phototransduction adaptation.

For over a decade, phosducin's interaction with the betagamma subunits of the G protein, transducin, has been thought to contribute to light adaptation by dynamically controlling the amount of transducin heterotrimer available for activation by photoexcited rhodopsin. In this study we directly tested this hypothesis by characterizing the dark- and light-adapted response properties of phosducin knockout (Pd- / -) rods. Pd- / - rods were notably less sensitive to light than wild-type (WT) rods. The gain of transduction, as measured by the amplification constant using the Lamb-Pugh model of activation, was 32% lower in Pd- / - rods than in WT rods. This reduced amplification correlated with a 36% reduction in the level of transducin betagamma-subunit expression, and thus available heterotrimer in Pd- / - rods. However, commonly studied forms of light adaptation were normal in the absence of phosducin. Thus, phosducin does not appear to contribute to adaptation mechanisms of the outer segment by dynamically controlling heterotrimer availability, but rather is necessary for maintaining normal transducin expression and therefore normal flash sensitivity in rods

Phosducin Regulates the Expression of Transducin βγ Subunits in Rod Photoreceptors and Does Not Contribute to Phototransduction Adaptation

For over a decade, phosducin's interaction with the βγ subunits of the G protein, transducin, has been thought to contribute to light adaptation by dynamically controlling the amount of transducin heterotrimer available for activation by photoexcited rhodopsin. In this study we directly tested this hypothesis by characterizing the dark- and light-adapted response properties of phosducin knockout (Pd−/−) rods. Pd−/− rods were notably less sensitive to light than wild-type (WT) rods. The gain of transduction, as measured by the amplification constant using the Lamb-Pugh model of activation, was 32% lower in Pd−/− rods than in WT rods. This reduced amplification correlated with a 36% reduction in the level of transducin βγ-subunit expression, and thus available heterotrimer in Pd−/− rods. However, commonly studied forms of light adaptation were normal in the absence of phosducin. Thus, phosducin does not appear to contribute to adaptation mechanisms of the outer segment by dynamically controlling heterotrimer availability, but rather is necessary for maintaining normal transducin expression and therefore normal flash sensitivity in rods

The Tetraspanin Protein Peripherin-2 Forms a Complex with Melanoregulin, a Putative Membrane Fusion Regulator

Peripherin-2, the product of the rds gene, is a tetraspanin protein. In this study, we show that peripherin-2 forms a complex with melanoregulin (MREG), the product of the Mreg locus. Genetic studies suggest that MREG is involved in organelle biogenesis. In this study, we explore the role of this protein in processes associated with the formation of disk membranes, specialized organelles of photoreceptor rod cells. MREG antibodies were generated and found to be immunoreactive with a 28 kDa protein in retinal extracts, bovine OS, ARPE-19 cells, and rat RPE. MREG colocalized with peripherin-2 in WT (CB6F1/J) and in rds+/- retinas. Western blots of serial tangential sections confirmed the close association of these two proteins within the IS and basal outer segment of rods. Immunoprecipitation (IP) of OS extracts showed formation of a complex between MREG and peripherin-2-ROM-1 hetero-oligomers. This interaction was confirmed with pulldown analyses in which the GST-PerCter protein selectively pulled down His-MREG and His-MREG selectively pulled down PerCter. Biacore analysis using peptide inhibitors and per-2 truncation mutant studies allowed us to map the MREG binding site on per-2 to the last five residues of the C-terminus (Gln341-Gly346), and kinetic data predicted a KD of 80 nM for PerCter-MREG binding. Finally, the effect of MREG on photoreceptor specific membrane fusion was assayed using a disk-plasma membrane cell free assay. Preincubation of target membranes with MREG resulted in a dose-dependent inhibition of fusion with an IC50 in the submicromolar range. Collectively, these results suggest that this newly identified protein regulates peripherin-2 function. © 2007 American Chemical Society

Compartment-specific Phosphorylation of Phosducin in Rods Underlies Adaptation to Various Levels of Illumination *

Phosducin is a major phosphoprotein of rod photoreceptors that interacts with the G� � subunits of heterotrimeric G proteins in its dephosphorylated state. Light promotes dephosphorylation of phosducin; thus, it was proposed that phosducin plays a role in the light adaptation of G protein-mediated visual signaling. Different functions, such as regulation of protein levels and subcellular localization of heterotrimeric G proteins, transcriptional regulation, and modulation of synaptic transmission have also been proposed. Although the molecular basis of phosducin interaction with G proteins is well understood, the physiological significance of light-dependent phosphorylation of phosducin remains largely hypothetical. In this study we quantitatively analyzed light dependence, time course, and subcellular localization of two principal light-regulated phosphorylation sites of phosducin, serine 54 and 71. To obtain physiologicall