Jay E. Reeder, Ph.D., Ronald Wood, Ph.D, Alan Friedman, Ph.D., Edward Schwarz, Ph.D., Edward M. Messing, M.D., Robert Mayer, M.D.

Department of Pathology and Laboratory Medicine, Department of Urology, Department of Obstetrics and Gynecology, Department of Environmental Medicine, Department of Orthopedics, University of Rochester, Rochester, New York

Introduction and Objectives: Antiproliferative activity found in the urine of interstitial cystitis (IC) patients has been linked to a glycosylated nonapeptide, (antiproliferative factor, APF) with primary sequence matching that of the sixth transmembrane domain of the seven transmembrane receptor, frizzled-8 (FZD8). We propose that translation of FZD8 transcripts initiated at the methionine codon at amino acid position 530 results in processing of the protein product in the secretory pathway and cleavage of the APF peptide as a signal peptide in the endoplasmic reticulum.

Methods: The FZD8 protein sequence was analyzed using SignalP 3.0, neural network and hidden Markov models to predict signal peptide and cleavage sites for all possible in frame AUG translational initiation sites. Additional processing of the transcript as a possible cell membrane protein was investigated using TMHMM software. A fusion gene, 530FZD8-EGFP was constructed by joining the FZD8 coding sequence, from codon 530 to the putative termination codon, with the enhanced green fluorescent protein sequence in the pcDNA3 plasmid. UMUC3 cells, a urothelial cancer cell line were transfected with the expression plasmid. Expression of the fusion protein was visualized by live cell fluorescence microscopy as well as following fixation and counterstaining of DNA. Stably transfected cells were obtained by selection with geneticin.

Results: The SignalP program predicts that initiation of translation of FZD8 transcripts at position 530 will produce a protein with a signal peptide recognized and processed in the endoplasmic reticulum. Cleavage of the putative signal peptide is predicted between amino acids 549 and 550, corresponding to the carboxy terminal residue of the APF nonapeptide. TMHMM predicts that the 165 amino acid sequence from position 530 to the carboxy terminal residue of FZD8 could be inserted into the plasma membrane with the carboxy terminal on the internal surface. The 530FZD8-EGFP fusion protein distributed in a pattern consistent with ER/Golgi processing and stably transfected cells show green fluorescence on the cell membrane.

Conclusions: These preliminary results are consistent with our hypothesis that APF could be a product of an aberrant translation of a normal or aberrant FZD8 transcript. For an active APF to be produced a mechanism for glycosylation and an additional cleavage is presumably required. There remains the possibility that the abbreviated FZD8 protein inserted in the membrane could have biological activity that could contribute to the etiology of IC.

This work was supported by the NIDDK, U01 DK065255, and DK066123

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