Abstract
Purpose: :
Guanylate Cyclase 1 (GC1) is a single span integral membrane protein that localizes to rod photoreceptor outer segments. Although GC1 plays a key role in trafficking of membrane-associated phototransduction proteins to cone outer segments, it remains unknown how GC1 itself targets to the outer segments. GC1 may contain a ROS localization signal like rhodopsin and peripherin/rds. To identify the GC1 sorting signal, we generated transgenic Xenopus laevis expressing eGFP fusion proteins in rod photoreceptors. These consisted of eGFP fused to the C-terminal sequence of X. laevis rhodopsin (XOP0.8-eGFP-rhoCT44), in which the last five amino acids of rhodopsin containing the VXPX sorting sequence was replaced by candidate GC1 fragments.
Methods: :
Mouse GC1 cDNA fragments were generated from retina RNA by RT-PCR. Transgenic tadpoles expressing the fusion protein were generated by nuclear transplantation. The expression pattern of the GFP fusion proteins were examined and analyzed by immunocytochemistry and confocal microscopy.
Results: :
Constructs encoding the following fusion proteins were generated by ligation of progressively longer cDNA fragments encoding regions of the cytoplasmic domain of GC1 in-frame with the eGFP-rhoCT44del5 cDNA. GCct1 consisted of 85 amino acids of the C-terminus of GC1; GCct2 included this sequence and the catalytic domain (CD); GCct3 included the C-terminus, CD and dimerization domain (DD); GCct4 incorporated the entire cytoplasmic domain of GC1, including the kinase homology domain. GCct1 randomly localized to the ROS, rod inner segment (RIS) plasma membrane and synaptic terminus. In contrast, GCct2 (CD and C-terminus) was observed primarily in the RIS plasma membrane and was nearly absent from ROS. In rods expressing GCct3 (DD+CD+C-terminus), the distribution of the fusion protein in ROS and RIS membranes was similar to GCct1. GCct4 containing the entire cytoplasmic domain of GC1 demonstrated nearly exclusive targeting of the fusion protein to ROS in a majority of rods examined. However, at higher expression levels the fusion protein was detected at low levels in the RIS plasma membrane.
Conclusions: :
These results suggest that the kinase homology domain of GC1 may be involved in ROS targeting. In contrast to peripherin and rhodopsin, this targeting domain is not located in the distal C-terminus, but is closely apposed to the lipid bilayer.
Keywords: photoreceptors • signal transduction • degenerations/dystrophies