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.

Although many different mutations in humans and Drosophila cause retinal degeneration, in most cases, a molecular mechanism for the degeneration has not been found. We now demonstrate the existence of stable, persistent complexes between rhodopsin and its regulatory protein arrestin in several different retinal degeneration mutants. Elimination of these rhodopsin-arrestin complexes by removing either rhodopsin or arrestin rescues the degeneration phenotype. Furthermore, we show that the accumulation of these complexes triggers apoptotic cell death and that the observed retinal degeneration requires the endocytic machinery. This suggests that the endocytosis of rhodopsin-arrestin complexes is a molecular mechanism for the initiation of retinal degeneration. We propose that an identical mechanism may be responsible for the pathology found in a subset of human retinal degenerative disorders.

Five mouse a-tubulin isotypes are described, each distinguished by the presence of unique amino acid substitutions within the coding region. Most, though not all of these isotype-specific amino acids, are clustered at the carboxy terminus. One of the a-tubulin isotypes described is expressed exclusively in testis and is encoded by two closely related genes (Ma3 and Ma7) which have homologous 3' untranslated regions but which differ at multiple third codon positions and in their 5' untranslated regions. We show that a subfamily of a-tubulin genes encoding the same testis-specific isotype also exists in humans. Thus, we conclude that (i) the duplication event leading to a pair of genes encoding a testis-specific ca-tubulin isotype predated the mammalian radiation, and (ii) both members of the duplicated sequence have been maintained since species divergence. A second ot-tubulin gene, Mca6, is expressed ubiquitously at a low level, whereas a third gene, MaL4, is unique in that it does not encode a carboxy-terminal tyrosine residue. This gene yields two transcripts: a 1.8-kilobase (kb) mRNA that is abundant in muscle and a 2.4-kb mRNA that is abundant in testis. Whereas the 1.8-kb mRNA encodes a distinct a-tubulin isotype, the 2.4-kb mRNA is defective in that the methionine residue required for translational initiation is missing. Patterns of developmental expression of the various at-tubulin isotypes are presented. Our data support the view that individual tubulin isotypes are capable of conferring functional specificity on different kinds of microtubules.Microtubules are assembled from heterodimers of a-and ,B-tubulin together with microtubule-associated proteins. They function in a wide variety of ways in eucaryotic cells; for example, they have specific functions in the mitotic and meiotic spindle, in the centriole, in the manchette and flagellar axoneme of spermatozoa, in axonal transport in neurons, and in the marginal bands of platelets. Clearly, associated proteins such as kinesin (28) play a crucial role in conferring these and other specific functions on microtubules. Additionally, a-and f-tubulin proteins themselves show significant heterogeneity, and the tubulin isotypes described to date vary in their patterns of expression in an evolutionarily conserved manner (16). This heterogeneity in a-and ,-tubulins offers the potential of contributing to diversity of microtubule function in eucaryotic cells, either through differential polymerization of the various tubulin subunits, or by virtue of unique interaction(s) with associated proteins.In mammals, a-and ,B-tubulins are encoded by large multigene families (5). A significant fraction of the genes in these families are pseudogenes (5, 13), a fact that makes it difficult to distinguish functional from nonfunctional genomic tubulin sequences. To circumvent this problem and to study the important question of the tubulin repertoire of mammals, we decided to study expressed mouse tubulin genes at the mRNA level by exhaustive screening of cDNA libraries. We re...

Progressive retinal degeneration is the underlying feature of many human retinal dystrophies. Previous work using Drosophila as a model system and analysis of specific mutations in human rhodopsin have uncovered a connection between rhodopsin endocytosis and retinal degeneration. In these mutants, rhodopsin and its regulatory protein arrestin form stable complexes, and endocytosis of these complexes causes photoreceptor cell death. In this study we show that the internalized rhodopsin is not degraded in the lysosome but instead accumulates in the late endosomes. Using mutants that are defective in late endosome to lysosome trafficking, we were able to show that rhodopsin accumulates in endosomal compartments in these mutants and leads to light-dependent retinal degeneration. Moreover, we also show that in dying photoreceptors the internalized rhodopsin is not degraded but instead shows characteristics of insoluble proteins. Together these data implicate buildup of rhodopsin in the late endosomal system as a novel trigger of death of photoreceptor neurons.

Arrestins are regulatory proteins that participate in the termination of G protein-mediated signal transduction. The major arrestin in the Drosophila visual system, Arrestin 2 (Arr2), is phosphorylated in a lightdependent manner by a Ca 2؉ ͞calmodulin-dependent protein kinase and has been shown to be essential for the termination of the visual signaling cascade in vivo. Here, we report the isolation of nine alleles of the Drosophila photoreceptor cellspecific arr2 gene. Flies carrying each of these alleles underwent light-dependent retinal degeneration and displayed electrophysiological defects typical of previously identified arrestin mutants, including an allele encoding a protein that lacks the major Ca 2؉ ͞calmodulin-dependent protein kinase site. The phosphorylation mutant had very low levels of phosphorylation and lacked the light-dependent phosphorylation observed with wild-type Arr2. Interestingly, we found that the Arr2 phosphorylation mutant was still capable of binding to rhodopsin; however, it was unable to release from membranes once rhodopsin had converted back to its inactive form. This finding suggests that phosphorylation of arrestin is necessary for the release of arrestin from rhodopsin. We propose that the sequestering of arrestin to membranes is a possible mechanism for retinal disease associated with previously identified rhodopsin alleles in humans.