EXPRESSION OF ALTERNATIVELY SPLICED ISOFORMS OF THE PARATHYROID-HORMONE (PTH) PTH-RELATED PEPTIDE RECEPTOR MESSENGER-RNA IN HUMAN KIDNEY AND BONE-CELLS/

Using a PCR-based strategy, two variants of the PTH/PTH-related peptid e (PTH-rp) receptor mRNA were identified in human kidney, SaOS-2 human osteoblast cells, and rat bone that are produced by alternative splic ing of exons coding for the N-terminal portion of the receptor. In the S-N3-E2 isoform, the exon coding the signal peptide (S) is spliced to an alternative 3'-acceptor site, producing a product respecting the r eading frame, but in which the El exon is replaced by 12 amino acids d erived from the N3 intron. In the S-E2 isoform, in which the E1 exon i s deleted by cassette exclusion, the reading frame is changed, but a t runcated receptor may be produced by reinitiation of translation at an overlapping stop/start codon. After transfection of COS and Chinese h amster ovary cells with the originally described S-E1-E2 isoform and t he two splice variants, active transcription of PTH/PTH-rp receptor mR NA was detected by RT-PCR in all cases. Cell lines transfected with th e S-E1-E2 and S-N3-E2 isoforms displayed a 15- to 25-fold and 2- to 3- fold increase, respectively, in cAMP content after stimulation with 2. 4 x 10(-7) M human PTH(1-34), whereas cells transfected with the S-E2 isoform did not respond. PTH elicited an increase in intracellular cal cium only in cells transfected with the S-E1-E2 isoform. Studies evalu ating the surface expression of receptors using antihuman PTH/PTH-rp r eceptor antibodies and the ability of transfected cells to bind [I-125 ]PTH-rp indicated that the low or absent responses to PTH stimulation resulted, at least in part, from low surface expression of the S-N3-E2 and S-E2 isoforms. These studies support the conclusion that exon El is extremely important in promoting surface expression of the PTH/PTH- rp receptor but indicate that isoforms lacking this exon can retain th e ability to recognize PTH. The possible intracellular expression of t hese splice variants, which account for 15-20% of total PTH/PTH-rp rec eptor mRNA, needs to be evaluated.