Multicellular life relies on the presence of extracellular matrix to provide scaffolding for cells and tissue compartments. To provide communication between cells and tissues, a multitude of cell surface receptors are triggered by soluble ligands and components of the extracellular matrix. A large family of these receptors transmit signals through the use of an intrinsic tyrosine kinase function. The subgroup of discoidin domain receptors (DDRs) is distinguished from other members of the receptor tyrosine kinase family by a discoidin homology repeat in their extracellular domains that is also found in a variety of other transmembrane and secreted proteins. Sequence comparisons show that non-mammalian orthologs of DDRs exist: three closely related genes in Caenorhabditis and one in the sponge Geodia cydonium. Recently, various types of collagen have been identified as the ligands for the two mammalian discoidin domain receptor tyrosine kinases, DDR1 and DDR2. The binding of collagen to DDRs results in a delayed but sustained tyrosine kinase activation. Both receptors display several potential tyrosine phosphorylation sites that are able to relay the signal by interacting with cytoplasmic effector proteins. The potential cross-talk to other receptors for collagen and the clinical aspects of DDR function are discussed.

https://faseb.onlinelibrary.wiley.com/doi/pdf/10.1096/fasebj.13.9001.s77

Discoidin domain receptors: structural relations and functional implications

▪ Abstract The past decade has witnessed extraordinary progress in retinal disease gene identification, the analysis of animal and tissue culture models of disease processes, and the integration of this information with clinical observations and with retinal biochemistry and physiology. During this period over twenty retinal disease genes were identified and for many of these genes there are now significant insights into their role in disease. This review presents an overview of the basic and clinical biology of the retina, summarizes recent progress in understanding the molecular mechanisms of inherited retinal diseases, and offers an assessment of the role that genetics will play in the next phase of research in this area.

Molecular Genetics of Human Retinal Disease

purpose. To examine the biochemical properties, cell expression, and localization of RS1, the product of the gene responsible for X-linked juvenile retinoschisis.

methods. Rs1h mRNA expression was measured from the eyes of wild-type and rd/rd mice by Northern blot analysis and reverse transcription–polymerase chain reaction (RT-PCR). Specific antibodies raised against the N terminus of RS1 were used as probes to examine the properties and distribution of RS1 in retina, retinal cell cultures, and transfected COS-1 cells by Western blot analysis and immunofluorescence microscopy.

results. Rs1h mRNA expression was detected in the retina of postnatal day (P)11 and adult CD1 mice, but not homozygous rd/rd mice by Northern blot analysis. However, Rs1h expression was detected in rd/rd mice by RT-PCR. RS1 migrated as a single 24-kDa polypeptide under disulfide-reducing conditions and a larger complex (>95 kDa) under nonreducing conditions in the membrane fraction of retinal tissue homogenates and transfected COS-1 cells. RS1 antibodies specifically stained rod and cone photoreceptors and most bipolar cells, but not Muller cells, ganglion cells, or the inner limiting membrane of adult and developing retina as revealed in double-labeling studies. RS1 antibodies also labeled retinal bipolar cells of photoreceptorless mice and retinal bipolar cells grown in cell culture.

conclusions. RS1 is expressed and assembled in photoreceptors of the outer retina and bipolar cells of the inner retina as a disulfide-linked oligomeric protein complex. The secreted complex associates with the surface of these cells, where it may function as a cell adhesion protein to maintain the integrity of the central and peripheral retina.

Expression of X-linked retinoschisis protein RS1 in photoreceptor and bipolar cells.

X-linked retinoschisis: A clinical and molecular genetic review

PURPOSE. To create and evaluate a mouse model of human X-linked juvenile retinoschisis (XLRS) and then investigate whether supplementing with the retinoschisin protein by gene delivery can reverse the abnormal “electronegative” electroretinogram (ERG) retinal response. METHODS. An X-linked retinoschisis mouse (Rs1h-KO) model was created by substituting a neomycin resistance cassette for exon 1 and 1.6 kb of intron 1 of Rs1h, the murine orthologue of the human RS-1 gene. RS protein was evaluated by immunohistochemistry and Western blot analysis with a polyclonal RS N-terminus antibody. Retinal function was evaluated by conventional, full-field flash ERG recordings. RS protein supplementation therapy was evaluated by gene transfer with an AAV(2/2)-CMV-Rs1h vector containing C57BL/6J Rs1h cDNA under the regulation of a CMV promoter, and ERG functional analysis was performed. RESULTS. No RS protein was detected by Western blot analysis or immunohistochemistry in the Rs1h-KO mouse. Darkadapted ERG responses showed an electronegative configuration, with b-wave reduction in both Rs1h /Y and Rs1h / mice, typical of XLRS in humans. Histologic examination of Rs1h-KO mice showed disorganization of multiple retinal layers, including duplication and mislocalization of ganglion cells, laminar dissection through the inner plexiform layer, disorganization of the outer plexiform layer, loss of regularity of the outer nuclear layer, and shortening of the inner/outer segments with mislocalization of photoreceptor nuclei into this layer. After intraocular administration of AAV(2/2)-CMV-Rs1h, immunohistochemistry showed retinoschisin expression in all retinal layers of Rs1h /Y mice, and ERG recordings showed reversal of the electronegative waveform and restoration of the normal positive b-wave. CONCLUSIONS. The RS-KO mouse mimics structural features of human X-linked juvenile retinoschisis with dissection through, and disorganization of, multiple retinal layers. The Rs1h-KO functional deficit results in an electronegative ERG waveform that is characteristic of human retinoschisis disease and that implicates a synaptic transmission deficit in the absence of retinoschisin protein. Replacement therapy by supplementing normal Rs1h protein in the adult Rs1h-KO mouse restored the normal ERG configuration. This indicates that gene therapy is a viable strategy of therapeutic intervention even in the postdevelopmental adult stage of XLRS disease. (Invest Ophthalmol Vis Sci. 2004;45:3279 –3285) DOI:10.1167/iovs.04-0576

RS-1 Gene Delivery to an Adult Rs1h Knockout Mouse Model Restores ERG b-Wave with Reversal of the Electronegative Waveform of X-Linked Retinoschisis.

X-linked retinoschisis (XLRS) is the most common cause of juvenile macular degeneration in males, resulting in vision loss early in life The gene involved in XLRS was identified recently It encodes a protein with a disoidin domain, suggested to be involved in cell-cell interactions We have screened the gene for mutations in 234 familial and sporadic retinoschisis cases and identified 82 different mutations in 214 (91%) Thirty one mutations were found more than once, ie 2-10 times, with the exception of the 214G→A mutation which was found in 34 apparently unrelated cases The origin of the patients, the linkage data and the site of the mutations (mainly CG dinucleotides) indicate that most recurrent mutations had independent origins and thus suggest the existence of a significant new mutation rate in XLRS1 The mutations identified cover the entire spectrum, from small intra-genic deletions (7%), to nonsense (6%), missense (75%), small frameshifting insertions/deletions (6%) and splice site mutations (6%) Since, regardless of the mutation type, no females with a typical RS phenotype were identified, RS seems to be caused by loss-of-function mutations only Mutations occurred non-randomly, with hotspots at several CG dinudeotides and a C6 stretch Exons 1-3 contained few, mainly translation-truncating mutations, arguing against an important functional role for this segment of the protein Exons 4-6, encoding the discoidin domain, contained most, mainly missense mutations An alignment of 32 discoidin domain proteins was constructed to reveal the consensus sequence and to deduce the functional importance of the missense mutations identified The mutation analysis revealed a high preponderance of mutations involving or creating cysteine residues, pointing to sites important for the tertiary folding and/or protein function, and highlights several amino acids which may be involved in XLRS1-specific protein-protein interactions Despite the enormous mutation heterogeneity, patients have relatively uniform clinical manifestations although with great intra-familial variation in age at onset and progression

V. Ventruto

Papers

Functional implications of the spectrum of mutations found in 234 cases with X-linked juvenile retinoschisis (XLRS)