Constitutive Activation of the Cyclic Adenosine 3′,5′-Monophosphate Signaling Pathway by Parathyroid Hormone (PTH)/PTH-Related Peptide Receptors Mutated at the Two Loci for Jansen’s Metaphyseal Chondrodysplasia

Abstract

Two different activating PTH/PTH-related peptide (PTHrP) receptor mutations, H223R and T410P, were recently identified as the most likely cause of Jansen's metaphyseal chondrodysplasia. To assess the functional importance of either amino acid position in the human PTH/PTHrP receptor, H223 and T410 were individually replaced by all other amino acids. At position 223, only arginine and lysine led to agonist-independent cAMP accumulation; all other amino acid substitutions resulted in receptor mutants that lacked constitutive activity or were uninformative due to poor cell surface expression. In contrast, most amino acid substitutions at position 410 conferred constitutive cAMP accumulation and affected PTH/PTHrP receptor expression not at all or only mildly. Mutations corresponding to the H223R or T410P exchange in the human PTH/PTHrP receptor also led to constitutive activity when introduced into the opossum receptor homolog, but showed little or no change in basal cAMP accumulation when introduced into the rat PTH/PTHrP receptor. The PTH/PTHrP receptor residues mutated in Jansen's disease are conserved in all mammalian members of this family of G protein-coupled receptors. However, when the equivalent of either the H223R or the T410P mutation was introduced into several other related receptors, including the PTH2 receptor and the receptors for calcitonin, secretin, GH-releasing hormone, glucagon-like peptide I, and CRH, the resulting mutants failed to induce constitutive activity. These studies suggest that two residues in the human PTH/PTHrP receptor, 223 and 410, have critical roles in signal transduction, but with different sequence constrains.

Constitutive activation of tethered-peptide/ corticotropin-releasing factor receptor chimeras

“…3b). Using an ELISA with antibodies raised against CRF(1-21) and CRF , the level of expression of CRF (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)[L8A])͞R1 ⌬N was found to be similar to that of CRF(1-16)͞R1 ⌬N (data not shown). These results support the hypothesis that the constitutive activation of the CRF(1-16)͞R1 ⌬N chimera is produced by specific interactions between the tethered amino-terminal part of CRF and the body of the receptor and that the specificity is reminiscent of that between CRF and native R1 (Fig.…”

“…The comparable mutant versions of the receptors for glucagon-like peptide I, gastric inhibitory peptide, calcitonin, secretin, growth hormone-releasing hormone, as well as for CRF fail to show ligand-independent activity (8). Even more surprisingly, the Thr-410-Pro point mutant in the rat PTH receptor also fails to produce ligand-independent activity (8).…”

“…Thus, it generally is believed that this region of the receptor plays a critical role in constraining the receptor in an inactive conformation. In the human PTH receptor, numerous mutations of the conserved Pro-410 induce constitutive activity (8). Therefore, this area may be similarly important for constraining the human PTH receptor in the inactive conformation.…”

“…Introduction of the His-223-Arg and Thr-410-Pro mutations at equivalent positions in the other secretin-like receptors results in a constitutively active phenotype for only the glucagon and vasoactive intestinal polypeptide receptors (8,13). The comparable mutant versions of the receptors for glucagon-like peptide I, gastric inhibitory peptide, calcitonin, secretin, growth hormone-releasing hormone, as well as for CRF fail to show ligand-independent activity (8).…”

β-Arrestin-dependent, G Protein-independent ERK1/2 Activation by the β2 Adrenergic Receptor

“…These were the starting point for the rational design of the TYY mutant described below. Four of these were eliminated, because mutational data either: linked them to increased G protein activation (Leu-275) (23)(24)(25)(26) or to decreased sequestration (Tyr-326) (27,28) or was insufficient (Asn-330 and Ala-134). By contrast, the remaining three residues had mutations shown to decrease G protein signaling in multiple receptors and with no data suggesting a decrease in internalization.…”