Novel functions of photoreceptor guanylate cyclases revealed by targeted deletion

Abstract

Targeted deletion of membrane guanylate cyclases (GCs) has yielded new information concerning their function. Here, we summarize briefly recent results of laboratory generated non-photoreceptor GC knockouts characterized by complex phenotypes affecting the vasculature, heart, brain, kidney, and other tissues. The main emphasis of the review, however, addresses the two GCs expressed in retinal photoreceptors, termed GC-E and GC-F. Naturally occurring GC-E (GUCY2D) null alleles in human and chicken are associated with an early onset blinding disorder, termed "Leber congenital amaurosis type 1" (LCA-1), characterized by extinguished scotopic and photopic ERGs, and retina degeneration. In mouse, a GC-E null genotype produces a recessive cone dystrophy, while rods remain functional. Rod function is supported by the presence of GC-F (Gucy2f), a close relative of GC-E. Deletion of Gucy2f has very little effect on rod and cone physiology and survival. However, a GC-E/GC-F double knockout (GCdko) phenotypically resembles human LCA-1 with extinguished ERGs and rod/cone degeneration. In GCdko rods, PDE6 and GCAPs are absent in outer segments. In contrast, GC-E(-/-) cones lack proteins of the entire phototransduction cascade. These results suggest that GC-E may participate in transport of peripheral membrane proteins from the endoplasmic reticulum (ER) to the outer segments.

Figure 1

Membrane guanylate cyclase knockout representations. White boxes, noncoding exons; black boxes or vertical lines, coding exons. TM, transmembrane domain. Neo cassettes indicate strategies for knockout constructs. References for knockout mice are: Npr1 [9]; Npr2 [11]; Gucy2c [97]; Gucy2d [18]; Gucy2e [82]; Gucy2f [31]; Gucy2g [21].

Figure 2

Distribution of GC1 and GC2 in WT (A,C) and GC knockout (B,D) mouse retina. Cryosections A,B were probed with anti-GC1, and C,D with anti-GC2 antibodies. GC1 is present in rod (intense green staining in the OS area) and cone (arrows) outer segments (A). GC2 is only detectable in WT rod outer segments (C). GC1 and GC2 are undetectable in the ONL or OPL (synaptic terminals). Faint immunofluorescence in the INL is nonspecific.

Figure 3

The rd chicken gene defect. Top, digram of the mutant gene in which exons 4-7 are replaced by an inverted fragment of exon 9 (shaded). Bottom, predicted WT gene representing exons 3-8. Exon 7a is an additional exon not present in mammalian GC-E genes. Adapted from [75].

Figure 4

Scotopic and photopic ERGs of WT, Gucy2e^-/-, Gucy2f^-/- and GCdko mice at 2.8 log cd s m^-2. Note lack of response in GCdko scotopic ERGs and in Gucy2e^-/- and GCdko photopic ERGs.

Figure 5

Distribution of phototransduction polypeptides in WT (A,C,E,G,I,K,M,O) and GCdko (B,D,F,H,J,L,N,P) mutant outer and inner segments. Antigens targeted by antibodies (green) are indicated top right in the GCdko panels. Nuclei of the ONL are counterstained with propidium iodide (red). The ‘patchy’ appearance of ROS indicates disorganization of the membrane structure [31].

Figure 6

Putative model of GC and rhodopsin transport following synthesis at the ER. A, WT rod photoreceptor. B, GCdko photoreceptor. In A, integral membrane proteins traffic from the ER to the Golgi/TGN by retrograde transport. Vesicles emerge from the TGN and are charged with the peripheral membrane proteins PDE and GCAPs. Retrograde transport continues to the base of the cilium. In B, since GC-E and GC-F are not produced, GCAPs and PDE cannot traffic, and are degraded. Rhodopsin, transducin, and GRK1 transport is not affected. For details, see text.

Figure 6

Putative model of GC and rhodopsin transport following synthesis at the ER. A, WT rod photoreceptor. B, GCdko photoreceptor. In A, integral membrane proteins traffic from the ER to the Golgi/TGN by retrograde transport. Vesicles emerge from the TGN and are charged with the peripheral membrane proteins PDE and GCAPs. Retrograde transport continues to the base of the cilium. In B, since GC-E and GC-F are not produced, GCAPs and PDE cannot traffic, and are degraded. Rhodopsin, transducin, and GRK1 transport is not affected. For details, see text.

Figure 7

Distribution of cone phototransduction polypeptides in WT (A,C,E,G,I,K,M,O) and GC1-/- (B,D,F,H,J,L,N,P) mutant outer and inner segments. Antigens targeted by antibodies (green) are indicated top right in the GCdko panels. Arrows (B,D) indicate disconnected membrane packets containing pigments. Visual pigments, cone transducin, cone PDE, GCAP1 and GRK1 are undetectable in mutant COS.

Figure 8

Putative model for transport of peripheral membrane proteins in cones. A, WT cone photoreceptor. B, GC-E knockout cone photoreceptor. In A, visual pigments and GC-E traffic from the ER to the Golgi/TGN by retrograde transport along microtubules. Vesicles emerge from the TGN and are charged with the peripheral membrane proteins PDEα′, cone T, GRK1, and GCAPs. Retrograde transport continues to the base of the cilium, followed by IFT to the outer segment. In B, GC-E is not produced. Peripheral proteins remain in the ER owing the absence of GC-E and are degraded.

Figure 8

Putative model for transport of peripheral membrane proteins in cones. A, WT cone photoreceptor. B, GC-E knockout cone photoreceptor. In A, visual pigments and GC-E traffic from the ER to the Golgi/TGN by retrograde transport along microtubules. Vesicles emerge from the TGN and are charged with the peripheral membrane proteins PDEα′, cone T, GRK1, and GCAPs. Retrograde transport continues to the base of the cilium, followed by IFT to the outer segment. In B, GC-E is not produced. Peripheral proteins remain in the ER owing the absence of GC-E and are degraded.