The structural basis for red fluorescence in the tetrameric GFP homolog DsRed.

Green fluorescent protein (GFP) has rapidly become a standard tool for investigating a variety of cellular activities, and has served as a model system for understanding spectral tuning in chromophoric proteins. Distant homologs of GFP in reef coral and anemone display two new properties of the fluorescent protein family: dramatically red-shifted spectra, and oligomerization to form tetramers. We now report the 1.9 A crystal structure of DsRed, a red fluorescent protein from Discosoma coral. DsRed monomers show similar topology to GFP, but additional chemical modification to the chromophore extends the conjugated pi-system and likely accounts for the red-shifted spectra. Oligomerization of DsRed occurs at two chemically distinct protein interfaces to assemble the tetramer. The DsRed structure reveals the chemical basis for the functional properties of red fluorescent proteins and provides the basis for rational engineering of this subfamily of GFP homologs.

A molecular pathway for light-dependent photoreceptor apoptosis in Drosophila.

Light-induced photoreceptor apoptosis occurs in many forms of inherited retinal degeneration resulting in blindness in both vertebrates and invertebrates. Though mutations in several photoreceptor signaling proteins have been implicated in triggering this process, the molecular events relating light activation of rhodopsin to photoreceptor death are yet unclear. Here, we uncover a pathway by which activation of rhodopsin in Drosophila mediates apoptosis through a G protein-independent mechanism. This process involves the formation of membrane complexes of phosphorylated, activated rhodopsin and its inhibitory protein arrestin, and subsequent clathrin-dependent endocytosis of these complexes into a cytoplasmic compartment. Together, these data define the proapoptotic molecules in Drosophila photoreceptors and indicate a novel signaling pathway for light-activated rhodopsin molecules in control of photoreceptor viability.

A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade.

How are signalling molecules organized into different pathways within the same cell? In Drosophila, the inaD gene encodes a protein consisting of five PDZ domains which serves as a scaffold to assemble different components of the phototransduction cascade, including the principal light-activated ion channels, the effector phospholipase C-beta and protein kinase C. Null inaD mutants have a dramatically reorganized subcellular distribution of signalling molecules, and a total loss of transduction complexes. Also, mutants defective in a single PDZ domain produce signalling complexes that lack the target protein and display corresponding defects in their physiology. A picture emerges of a highly organized unit of signalling, a 'transduclisome', with PDZ domains functioning as key elements in the organization of transduction complexes in vivo.

Regulation of PLC-mediated signalling in vivo by CDP-diacylglycerol synthase.

CDP-diacylglycerol synthase (CDS) is an enzyme required for the regeneration of the signalling molecule phosphatidylinositol-4,5-bisphosphate (PtdlnsP2) from phosphatidic acid. A photo-receptor cell-specific isoform of CDS from Drosophila is a key regulator of phototransduction, a G-protein-coupled signalling cascade mediated by phospholipase C. cds mutants cannot sustain a light-activated current as a result of depletion of PtdlnsP2. Overexpression of CDS increases the amplitude of the light response, demonstrating that availability of PtdlnsP2 is a determinant in the gain of this pathway. cds mutants undergo light-dependent retinal degeneration which can be suppressed by a mutation in phospholipase C. Thus, enzymes involved in PtdlnsP2 metabolism regulate phosphoinositide-mediated signalling cascades in vivo.

Arrestin function in inactivation of G protein-coupled receptor rhodopsin in vivo.

Arrestins have been implicated in the regulation of many G protein-coupled receptor signaling cascades. Mutations in two Drosophila photoreceptor-specific arrestin genes, arrestin 1 and arrestin 2, were generated. Analysis of the light response in these mutants shows that the Arr1 and Arr2 proteins are mediators of rhodopsin inactivation and are essential for the termination of the phototransduction cascade in vivo. The saturation of arrestin function by an excess of activated rhodopsin is responsible for a continuously activated state of the photoreceptors known as the prolonged depolarized afterpotential. In the absence of arrestins, photoreceptors undergo light-dependent retinal degeneration as a result of the continued activity of the phototransduction cascade. These results demonstrate the fundamental requirement for members of the arrestin protein family in the regulation of G protein-coupled receptors and signaling cascades in vivo.

Ectopic expression of ultraviolet-rhodopsins in the blue photoreceptor cells of Drosophila: visual physiology and photochemistry of transgenic animals.

We have generated transgenic flies expressing R7 cell-specific opsins in the major class of photoreceptor cells of the Drosophila retina and characterized their spectral properties using high-resolution microspectrophotometry and sensitivity recordings. We show that the Rh3 and Rh4 opsin genes encode UV-sensitive opsins with similar spectral properties (lambda max = 345 nm and 375 nm), and that Rh3 corresponds to the R7p and R7marg class of visual pigments. We have also generated Rh3 and Rh4 isoform-specific antibodies and present an R7 cell map of the Drosophila retina. In a related set of experiments, we show that it is possible to coexpress two different visual pigments functionally in the same cell and produce photoreceptors that display the summed spectral response of the individual pigments. These findings open up the possibility of tuning an animal's visual behavior by targeted expression of combinations of opsin genes to selective types of photoreceptors.