TISSUE-SPECIFIC TRANSCRIPTION START SITES AND ALTERNATIVE SPLICING OFTHE PARATHYROID-HORMONE (PTH) PTH-RELATED PEPTIDE (PTHRP) RECEPTOR GENE - A NEW PTH/PTHRP RECEPTOR SPLICE VARIANT THAT LACKS THE SIGNAL PEPTIDE/

The PTH/PTHrP receptor gene is expressed in bone and kidney as well as in many other tissues. Using primer extension followed by rapid cloni ng of amplified complementary DNA ends, we have isolated new PTH/PTHrP receptor complementary DNAs with different splicing patterns and have characterized a new upstream transcription start site. Three 5' nontr anslated exons, U3, U2 and U1, located 4.8, 2.5, and 1.2 kb upstream o f the exon that encodes the putative signal peptide of the classical r eceptor (exon S), have been characterized. Four types of splicing patt erns were recognized. Type I splicing pattern is transcribed from exon U1 and is spliced to exons S and E1; this pattern was found in most t issues tested. Types II, III, and IV splicing patterns are transcribed from exon U3 and have a restricted tissue distribution. Type II splic e pattern, containing exons U3, U2, and S and type III splicing patter n, containing exon U3, U2, and El (skipping exon S), was found only in kidney. Type IV splice pattern, containing exon U3 and S was found bo th in kidney and ovary. Because the type III splice variant skips exon S, translation of this splice Variant initiates at a different AUG co don. The type III splice variant was weakly expressed on the cell surf ace of COS-7 cells, as assessed by double antibody binding assay, and no detectable ligand binding was observed on intact cells. The type II I splice variant, however, increased cAMP accumulation in COS-7 cells when challenged with PTH(1-34), PTH(1-84) and hPTHrP(1-36) with EC50s that are similar to those observed in COS-7 cells expressing the type I variant but with a maximum stimulation that was lower than that obse rved in COS-7 cells expressing the type I variant. These data indicate low levels of cell surface expression of the type III splice variant. Treatment of COS-7 cells with tunicamycin decreased the size of the t ype I splice variant from a broad band of 85 kDa to a compact band of about 60 kDa. The type III splice variant did not change in size in CO S-7 cells treated with tunicamycin, indicating that the type III splic e variant did not undergo any glycosylation step. In conclusion, the P TH/PTHrP receptor gene uses alternate promoters in a tissue-specific m anner that results in several tissue-specific alternatively spliced tr anscripts. One of these transcripts, the type III splice variant, is e xpressed in kidney and lacks the signal peptide.