Ligand binding and signalling pathways of PTH receptors in sea bream (Sparus auratus) enterocytes.
Whole animal studies have indicated that Ca(2+) uptake by the gastrointestinal tract is regulated by the action of parathyroid hormone-related peptide (PTHrP) in teleost fish. We have characterised PTH receptors (PTHR) in piscine enterocytes and established, by using amino-terminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of (125)I-(1-35(tyr)) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (K (D)=2.59 nM; B (max)=71 fmol/mg protein). Reverse transcription/polymerase chain reaction with specific primers for sea bream PTH1R and PTH3R confirmed the mRNA expression of only the later receptor. Fugu (1-34)PTHrP increased cAMP levels in enterocytes but had no effect on total inositol phosphate accumulation. The amino-terminal peptides (2-34)PTHrP, (3-34)PTHrP and (7-34)PTHrP bound efficiently to the receptor but were severely defective in stimulating cAMP in enterocyte cells indicating that the first six residues of piscine (1-34)PTHrP, although not important for receptor binding, are essential for activation of the adenylate cyclase/phosphokinase A (AC-PKA)-receptor-coupled intracellular signalling pathway. Therefore, PTHrP in teleosts acts on the gastrointestinal tract through PTH3R and the AC-PKA intracellular signalling pathway and might regulate Ca(2+) uptake at this site. Ligand-receptor binding and activity throughout the vertebrates appears to be allocated to the same amino acid residues of the amino-terminal domain of the PTHrP molecule.
Rotllant
J
J
Centre of Marine Sciences, CIMAR-Laboratório Associado, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal. rotllant@umbi.umd.edu
Guerreiro
P M
PM
Redruello
B
B
Fernandes
H
H
Apolónia
L
L
Anjos
L
L
Canario
A V M
AV
Power
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020013:Calcium Signaling; D000242:Cyclic AMP / Q000378:metabolism; D020895:Enterocytes / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D008024:Ligands; D008969:Molecular Sequence Data; D044162:Parathyroid Hormone-Related Protein / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021541:Sea Bream / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D023741:Takifugu / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics
D008024:Ligands; D044162:Parathyroid Hormone-Related Protein; D012333:RNA, Messenger; D044168:Receptor, Parathyroid Hormone, Type 1; D000242:Cyclic AMP