A Potential Interaction Between the RP1 and IMPDH1 Proteins

Abstract

Abstract: : Purpose: Mutations in the RP1 gene are a common cause of adRP. The mechanism by which mutations in RP1 lead to photoreceptor death and the function of RP1 protein are only starting to become understood. To help elucidate the function of RP1 protein, we used co–immunoprecipitation (co–IP) experiments to identify RP1 interacting proteins. Methods: Total mouse retinal protein extracts were incubated with anti–Rp1 antibodies, followed by precipitation with Protein A beads. A Coomasie blue stained SDS–PAGE gel of the precipitated proteins identified a 64kD candidate interacting protein. This 64 kD band was subjected to mass spectrometric analysis. IMPDH1 cDNAs amplified from retinal RNA by RT–PCR were cloned into expression vectors with Xpress or Flag tags. The N–terminal portion of RP1(N–RP1) and IMPDH1 expression plasmids were co–transfected into 293 cells and co–IP was performed using anti–tag antibodies. Similar co–IP experiments were performed using mutant IMPDH1 constructs. Immunostaining of mouse retina was performed using anti–IMPDH1 and Rp1 antibodies. Results: Mass spectrometric analysis identified the 64kD protein as mouse Impdh1, the product of the RP10 disease gene. This result was confirmed by immunoblotting Rp1–precipiated proteins with Impdh1 antibodies. Cloning of the human and mouse IMPDH1 cDNAs from retina revealed two extra coding regions: one (147bp) from the 5’ non–coding exon A and one (17bp) from the last intron, which causes a frame shift that adds 79bp of coding sequence at the 3' end. The MW of the proteins predicted to be produced from these longer retinal IMPDH1 transcripts is 64 kD, compared to the 55 kD reported for IMPDH1 from other tissues. Co–IP of N–RP1 and 64kD– or 55kD–IMPDH1 recombinant proteins showed that both 64kD and 55kD IMPDH1 are precipitated by N–RP1. In contrast, mutant IMPDH1 proteins were not efficiently precipitated by N–RP1. Immunostaining of mouse retinas with anti–Impdh1 antibodies showed the strongest signal in the photoreceptor inner segments. Conclusions: We have identify IMPDH1 as a candidate RP1 interacting protein. This interaction was disrupted when disease causing mutations were introduced into IMPDH1. This result suggests that RP1 and IMPDH1 may participate in a common biochemical pathway, and that RP1 and RP10 diseases may share important pathogenetic features. Further in vitro and in vivo experiments are in progress to verify this RP1/IMPDH1 interaction and to define the functional significance of the interaction. NIH (EY12910), RPB, FFB, Rosanne Silbermann Foundation

Keywords: photoreceptors • retinal degenerations: cell biology • proteins encoded by disease genes