The function of oligomerization-incompetent RDS in rods

Abstract

The photoreceptor-specific tetraspanin glycoprotein RDS (retinal degeneration slow) is associated with many forms of inherited retinal disease. RDS shares features in common with other tetraspanin proteins, including the existence of a large intradiscal D2 loop containing several cysteines. While these cysteines are used only for intramolecular disulfide bonds in most tetraspanins, RDS expresses a seventh, unpaired cysteine (C150) used for intermolecular disulfide bonding in the formation of large RDS oligomers. To study oligomerization-dependent vs. oligomerization-independent RDS functions in rods, we generated a transgenic mouse line harboring a point mutation that replaces this Cys with Ser (C150S), leading to the expression of an RDS protein that cannot form intermolecular disulfide bonds. The mouse opsin promoter (MOP) was used to direct C150S RDS expression specifically in rods in these transgenic mice (MOP-T). Here we report improvement in scotopic ERGs in MOP-T/rds ( +/- ) mice (compared to non-transgenic rds ( +/- ) controls) and the appearance of malformed outer segments (OSs) in MOP-T mice that do not express native RDS (MOP-T/rds ( -/- )). These results suggest that while normal OS structure and function require RDS oligomerization, some RDS function is retained in the absence of C150. Since one of the functions of other tetraspanin proteins is to promote assembly of a membrane microdomain known as the "tetraspanin web", future studies may investigate whether assembly of this web is one of RDS's oligomerization-independent functions.

Fig. 5.1

Biochemical analysis of MOP-T; Retinas were harvested from transgenic (MOP-T) or non-transgenic (NT) animals at postnatal day (P) 30. Total retinal extracts were used for non-reducing (a) or reducing (b) SDS-PAGE followed by immunoblotting (IB) with the antibodies indicated (RDS-CT, ROM-1, rod outer segment membrane protein-1, an RDS homolog; and actin). Expression of C150S RDS does not interfere with the ability of WT RDS to form higher-order oligomers, but it cannot form oligomers alone

Fig. 5.2

Functional analysis of MOP-T; (a) Non-transgenic (WT) and transgenic (MOP-T/WT) animals in the WT background underwent full-field scotopic ERG. Shown are maximum scotopic a-wave amplitudes at various ages as indicated. At both early (1–2 months) and late (18 months) ages, there is no difference in maximum scotopic ERG amplitude between transgenic and non-transgenic mice. (b) Toluidine blue stained, plastic embedded sections were collected from eyes harvested from transgenic and non-transgenic animals at 18 months of age. Scale bar, 20 μm. The C150S transgene has no structural effect on the retina in the WT background. (c) At P60, transgenic and non-transgenic animals in multiple RDS backgrounds underwent full-field scotopic and photopic ERG. Shown are averages (+/− S.E.M.) from 5 to 7 animals/category. In the absence of WT RDS, no retinal function is detected, however, the presence of the C150S transgene improves the maximum scotopic a-wave amplitude in the heterozygous rds^+/− mouse

Fig. 5.3

Ultrastructural analysis of MOP-T; Eyes were harvested from P30 transgenic and non-transgenic eyes and processed for immunogold/EM (a) or EM (b). (a) mAB 1D4 against rhodopsin was used to label OSs in MOP-T/rds^{−/ −} and WT (as a control) animals. No normal OSs were observed in MOP-T/rds^{−/ −} animals, only rounded whorl-like membranous structures reminiscent of those seen in the rds^+/− (b), only larger. Scale bar, 2 μm