Abstract
Purpose: :
It is unknown how guanylate cyclase 1 (GC1) targets to the outer segments where it resides. To identify a putative GC1 targeting signal, we generated a series of peripheral membrane (PM) and transmembrane (TM) constructs encoding extracellular and intracellular mouse GC1 fragments fused to eGFP for expression in X. laevis rod photoreceptors.
Methods: :
Mouse GC1 cDNA fragments were generated from retinal RNA by RT-PCR. For PM constructs, eGFP and cytoplasmic GC1 sequences were ligated to the C-terminus of X. laevis rhodopsin lacking the rhodopsin targeting signal (eGFP-rhoCT44del5). For TM constructs, the extracellular or cytoplasmic sequences of GC1 were replaced by eGFP. Transgenic tadpoles expressing the eGFP fusion proteins were generated by nuclear transplantation. Subcellular localization of the GFP fusion proteins were analyzed by immunocytochemistry and confocal microscopy.
Results: :
GC1 consists of an extracellular domain (ECD), a transmembrane domain (TM), a kinase-like homology domain (KHD), a dimerization domain (DD), and a catalytic domain (CAT). Eight PM fusion proteins (GCct1-GCct8) contained combinations of the immediate C-terminus, CAT, DD, or KHD. Additionally, four TM constructs (GCtm9-12) consisted of full length GC1 fused to eGFP (GCtm10); in GCtm9 the ECD was replaced by eGFP; in GCtm11 the cytoplasmic domain was replaced by eGFP, and in GCtm12 the rhodopsin targeting signal TETSQVAPA was added to the C-terminus of GCtm9. Of the eight PM fusions, none targeted perfectly to the outer segments (OS), seven showed significant mislocalization to the inner segment (IS) and synapse, only one containing the entire cytoplasmic domain targeted primarily to OS. Three fusion proteins containing either C-term+CAT, KHD or KHD+DD, were excluded from the OS. Of the TM fusion proteins, GCtm10 and GCtm9 showed near perfect targeting to the OS while GCtm11 mistargeted to the OS, IS and synapse. Addition of TETSQVAPA in GCtm12 did not prevent mistargeting in some rods, presumably because of overexpression/misfolding of the fusion protein. As a group, fusion proteins containing the entire cytoplasmic domain of GC1 (GCct4, GCtm9, GCtm10 and GCtm12) targeted to the OS nearly correctly.
Conclusions: :
GC1 likely has no single linear peptide-based OS targeting signal in the cytoplasmic or extracellular domains. Our results suggest targeting is due to either multiple weak signals in the cytoplasmic domain of GC1, or co-transport to the OS with another protein.
Keywords: photoreceptors • signal transduction • transgenics/knock-outs