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 peptide (PTH-rp) receptor mRNA were identified in human kidney, SaOS-2 human osteoblast cells, and rat bone that are produced by alternative splicing 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 reading frame, but in which the E1 exon is replaced by 12 amino acids derived from the N3 intron. In the S-E2 isoform, in which the E1 exon is deleted by cassette exclusion, the reading frame is changed, but a truncated receptor may be produced by reinitiation of translation at an overlapping stop/start codon. After transfection of COS and Chinese hamster ovary cells with the originally described S-E1-E2 isoform and the two splice variants, active transcription of PTH/PTH-rp receptor mRNA was detected by RT-PCR in all cases. Cell lines transfected with the 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 calcium only in cells transfected with the S-E1-E2 isoform. Studies evaluating the surface expression of receptors using anti-human PTH/PTH-rp receptor antibodies and the ability of transfected cells to bind [125I]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 E1 is extremely important in promoting surface expression of the PTH/PTH-rp receptor but indicate that isoforms lacking this exon can retain the ability to recognize PTH. The possible intracellular expression of these splice variants, which account for 15-20% of total PTH/PTH-rp receptor mRNA, needs to be evaluated.
Jobert
A S
AS
INSERM U 426, Faculté de Médecine Xavier Bichat, Paris, France.
Fernandes
I
I
Turner
G
G
Coureau
C
C
Prie
D
D
Nissenson
R A
RA
Friedlander
G
G
Silve
C
C
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001842:Bone and Bones / Q000378:metabolism*; D016466:CHO Cells / Q000378:metabolism; D019556:COS Cells / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry; D006801:Humans; D007457:Iodine Radioisotopes; D007668:Kidney / Q000378:metabolism*; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D018016:Receptors, Parathyroid Hormone / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D007457:Iodine Radioisotopes; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D012333:RNA, Messenger; D044168:Receptor, Parathyroid Hormone, Type 1; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; D000242:Cyclic AMP; D002118:Calcium