Interaction and localization of the retinitis pigmentosa protein RP2 and NSF in retinal photoreceptor cells

Abstract

RP2 is a ubiquitously expressed protein encoded by a gene associated with X-linked retinitis pigmentosa (XLRP), a retinal degenerative disease that causes severe vision loss. Previous in vitro studies have shown that RP2 binds to ADP ribosylation factor-like 3 (Arl3) and activates its intrinsic GTPase activity, but the function of RP2 in the retina, and in particular photoreceptor cells, remains unclear. To begin to define the role of RP2 in the retina and XLRP, we have conducted biochemical studies to identify proteins in retinal cell extracts that interact with RP2. Here, we show that RP2 interacts with N-ethylmaleimide sensitive factor (NSF) in retinal cells as well as cultured embryonic kidney (HEK293) cells by mass spectrometry-based proteomics and biochemical analysis. This interaction is mediated by the N-terminal domain of NSF. The E138G and DeltaI137 mutations of RP2 known to cause XLRP abolished the interaction of RP2 with the N-terminal domain of NSF. Immunofluorescence labeling studies further showed that RP2 colocalized with NSF in photoreceptors and other cells of the retina. Intense punctate staining of RP2 was observed close to the junction between the inner and outer segments beneath the connecting cilium, as well as within the synaptic region of rod and cone photoreceptors. Our studies indicate that RP2, in addition to serving as a regulator of Arl3, interacts with NSF, and this complex may play an important role in membrane protein trafficking in photoreceptors and other cells of the retina.

Figure 1

Characterization of RP2-6H2 antibody using bovine retinal extract and His-tagged human RP2. (A) Coomassie blue-stained gel (CB) and a western blot of a retinal extract (retina) and purified His-tagged RP2 (RP2) labeled with the RP2-6H2 antibody demonstrating the specificity of the antibody. (B) A series of GST-RP2 fusion proteins were used to map the epitope of RP2-6H2 antibody. The RP2-6H2 antibody labels the full length (GST-RP2) and the N-terminal fusion protein (GST-RP2-N) but not the C-terminal fusion protein (GST-RP2-C) or GST-RP2 lacking the first 34 amino acids (GST-RP2-Δ34) as shown in the right panel. The GST-fusion proteins were identified in the western blot labeled with an anti-GST antibody.

Figure 2

Immunofluorescence microscopy of mouse and human retinal cryosections labeled with the RP2-6H2 antibody. RP2 in both mouse and human retina is present throughout most of the retina, including intense labeling in the inner segment and outer plexiform layer of the outer retina and the inner nuclear layer, inner plexiform layer, and ganglion cell layer of the inner retina; labeling is absent in the outer segment layer. Inset: An enlarged image of the junction between the inner and outer segments showing punctate labeling of RP2. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bar = 20 μm.

Figure 3

Immunoprecipitation of retinal and HEK293 cell membrane extracts on a RP2-6H2 immunoaffinity matrix. (A) The solubilized retinal membranes (Input) and unbound (Unbound) and SDS-eluted (Bound) fractions were analyzed on SDS gels stained with Coomassie blue (CB) and western blots labeled with the RP2-6H2 antibody (RP2). Quantitative immunoprecipitation was observed for RP2. The SDS-eluted fraction from a control sample (Control) without retinal membranes revealed the immunoglobulin protein bands (*) that dissociated from the immunoaffinity matrix under denaturing conditions. The western blot labeled with the NSF antibody (NSF) confirmed co-immunoprecipitation of NSF with RP2 from retinal extract. (B) Western blots of retinal extracts labeled with antibodies against a and β tubulin and Arl3 showed that little if any of these proteins co-immunoprecipitated with RP2. (C) Endogenous NSF from HEK293 cell homogenate also co-immunoprecipitated with endogenous RP2.

Figure 4

RP2 is associated with the membrane fraction of a retinal extract. Western blots of retinal extracts with RP2-6H2 antibody and antibodies against NSF and Arl3 show that RP2 is preferentially present in the membrane fraction while NSF is present in both fractions and Arl3 is primarily in the soluble fraction.

Figure 5

Confocal scanning microscopy of mouse retina double labeled for RP2 and NSF. (A) Labeling of NSF with an anti-NSF polyclonal antibody; (B) labeling of RP2 with the RP2-6H2 monoclonal antibody; (C) merged image showing co-localization of RP2 and NSF. More intense co-staining was observed for both NSF and RP2 near the junction of the inner and outer segments and outer plexiform layers (D–F). In a number of samples, punctuate staining was observed for RP2 (H) and not NSF (G). Merged image (I) showing RP2 and NSF are not co-localized at these punctations. Bar = 20 μm. Retinal layers are defined in Figure 2.

Figure 6

Binding of RP2 to the N-terminal domain of NSF. (A) Coomassie Blue-stained gel showing the purified N-terminal (GST-NTNSF), D1 (GST-D1NSF), and D2 (GST-D2NSF) fusion proteins and the purified recombinant wild-type (WTRP2), and disease-causing mutant (E138G and L188P) RP2 proteins. (B) Western blot labeled with the RP2-6H2 antibody shows that RP2 interacts primarily with the fusion protein containing the N-terminal domain of NSF. GST protein was used as a control.

Figure 7

Binding of disease-linked mutants of RP2 to N-terminal domain of NSF. The binding of wild-type (WT) and mutant RP2 proteins to immobilized GST fusion protein containing the N-terminal domain of NSF. Binding was detected on western blots labeled with the RP2-6H2 antibody. The binding of RP2 to the N-terminal NSF domain is essentially abolished for the E138G and Δ137 RP2 disease mutations.