ROLE OF THE EXTRACELLULAR REGIONS OF THE PARATHYROID-HORMONE (PTH) PTH-RELATED PEPTIDE RECEPTOR IN HORMONE-BINDING

The PTH/PTH-related peptide receptor is a member of a newly discovered family of G-protein-coupled receptors. Strikingly conserved features among these receptors include the positioning of eight extracellular c ysteines and several other residues that are located predominantly wit hin the membrane-embedded region. Deletion mutants or receptors with p oint mutations of the highly conserved cysteine residues were transien tly expressed in COS-7 cells to evaluate PTH binding and PTH-stimulate d cAMP production. Deletion of residues 61-105, which are encoded by e xon E2 in the PTH/PTH-related peptide receptor gene, did not affect re ceptor function. An epitope derived from Haemophilus influenza hemaggl utinin was, therefore, introduced into this portion of most receptors to allow the independent assessment of cell surface expression. PTH bi nding capacity was not reduced by the deletion of residues 258 and 278 in the first extracellular loop. Receptors with deletion of either re sidues 31-47 in the amino-terminal extension or residues 431-440 in th e third extracellular loop failed to bind PTH, although expression of the receptor on the cell surface was only marginally reduced. Most oth er receptor mutants, including those in which each of the six cysteine s in the amino-terminus was replaced by serines, failed to be processe d and/or expressed appropriately, whereas the substitution of cysteine -281 or -351 had a less severe effect. The combined replacement of bot h cysteines concomitantly increased PTH binding and cell surface expre ssion, suggesting the formation of a disulfide bond between these two residues. Our data indicate that residues near the amino-terminus and within the third extracellular loop are necessary for ligand binding, whereas more than 25% of the receptor's extracellular region appears n ot to be involved.