Abstract
The functional role of the rat parathyroid hormone(PTH)/PTH-related peptide (PTHrP) receptor's carboxyl-terminal region was characterized by comparing the binding and signaling properties of receptors that have 78 and 111 amino acid deletions (R513 and R480, respectively), with those of the 591-amino acid wild-type (WT) receptor. R480 and R513 have 4- and 1.5-fold lower apparent Kd values for rat PTH-(1-34) (rPTH), compared with the WT receptor (WT, 1.81 +/- 0.19 nM; R513, 1.24 +/- 0.12 nM; R480, 0.48 +/- 0.05 nM, mean +/- S.E.). PTH (100 nM)-stimulated cAMP accumulation and polyphosphoinositide hydrolysis both correlated positively with receptor expression. However, whereas PTH-stimulated polyphosphoinositide hydrolysis was indistinguishable among WT and either truncated mutant at comparable levels of expressed receptors, maximal PTH-stimulated cAMP accumulation was 4-6- and 2-3-fold higher in cells expressing R480 and R513, respectively. Furthermore, pretreatment of COS-7 cells with 100 ng/ml of pertussis toxin (PTX) enhanced PTH-stimulated cAMP accumulation in cells expressing the WT receptor, but failed to do so in cells expressing either R480 or R513. Thus, sequences in the PTH/PTHrP receptor's carboxyl-terminal tail lower the affinity of the WT receptor for agonist; directly interact with, or indirectly facilitate the interaction of the receptor with a PTX-sensitive G protein that inhibits adenylyl cyclase; and decrease the efficacy with which the receptor interacts with Gs.
Highlights
IntroductionThe functional role of the rat parathyroid hormone(PTH)/PTH-related peptide (PTHrP) receptor's carboxyl-terminal region was characterized by comparing the binding and signaling properties of receptors that have 78 and Ill amino acid deletions (R513 and R480, respectively), with those of the 591-amino acid wild-type (WT) receptor
Our most striking observation is that rat parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptors with truncated carboxyl-terminal, intracellular regions signal adenylyl cyclase with markedly higher efficacy, compared with the WT receptor, but have indistinguishable capacities to hydrolyze Pl
We initially noted lower ligand binding in cells transfected with R480 and R513, compared with the WT receptor, when the same amount of DNA was used for transfection
Summary
The functional role of the rat parathyroid hormone(PTH)/PTH-related peptide (PTHrP) receptor's carboxyl-terminal region was characterized by comparing the binding and signaling properties of receptors that have 78 and Ill amino acid deletions (R513 and R480, respectively), with those of the 591-amino acid wild-type (WT) receptor. Whereas PTHstimulated polyphosphoinositide hydrolysis was indistinguishable among WT and either truncated mutant at comparable levels of expressed receptors, maximal PTH-stimulated cAMP accumulation was 4-6- and 2-3fold higher in cells expressing R480 and R513, respectively. The parathyroid hormone (PTH)1/PTH-related peptide (PTHrP) receptor (1, 2) belongs to an unique family within the seven membrane-spanning guanine-nucleotide regulatory protein (G protein)-coupled receptor superfamily. This family includes mammalian receptors for calcitonin (3), secretin (4),. An insect diuretic hormone receptor (13) and a partial genomic sequence from Caenorhabditis elegans (14) are homologous with the PTHIPTHrP receptor, indicating that this newly discovered family is widely conserved through evolution
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