Cell signaling mediated by the G protein-coupled parathyroid hormone receptor type 1 (PTHR) is fundamental to bone and kidney physiology. It has been unclear how the two ligand systems-PTH, endocrine and homeostatic, and PTH-related peptide (PTHrP), paracrine-can effectively operate with only one receptor and trigger different durations of the cAMP responses. Here we analyze the ligand response by measuring the kinetics of activation and deactivation for each individual reaction step along the PTHR signaling cascade. We found that during the time frame of G protein coupling and cAMP production, PTHrP 1-36 action was restricted to the cell surface, whereas PTH 1-34 had moved to internalized compartments where it remained associated with the PTHR and Gα s , potentially as a persistent and active ternary complex. Such marked differences suggest a mechanism by which PTH and PTHrP induce differential responses, and these results indicate that the central tenet that cAMP production originates exclusively at the cell membrane must be revised.Seminal studies during the past decades established that signaling cascades mediated by a G protein-coupled receptor (GPCR) initially proceed through a succession of biochemical events that take place at the cell membrane and result in the induction and propagation of second messenger molecules 1-4 (Fig. 1a). These events begin with the binding of an 'agonist' ligand (L) to an inactive-state receptor (R), which causes the receptor to switch to an active-state conformation (R*). The activated receptor then interacts with heterotrimeric G proteins (G, or Gαβγ) to form a transient L-R*-G complex, which exhibits higher affinity for the agonist ligand than does the initial L-R state. The interaction process further involves a conformational change-induced exchange of GDP for GTP on Gα with concomitant release of the activated, GTP-bound Gα (along with Gβγ) from the L-R complex, and the

The parathyroid hormone receptor-1 (PTH1R) is a class B G protein–coupled receptor central to calcium homeostasis and a therapeutic target for osteoporosis and hypoparathyroidism. Here we report the cryo–electron microscopy structure of human PTH1R bound to a long-acting PTH analog and the stimulatory G protein. The bound peptide adopts an extended helix with its amino terminus inserted deeply into the receptor transmembrane domain (TMD), which leads to partial unwinding of the carboxyl terminus of transmembrane helix 6 and induces a sharp kink at the middle of this helix to allow the receptor to couple with G protein. In contrast to a single TMD structure state, the extracellular domain adopts multiple conformations. These results provide insights into the structural basis and dynamics of PTH binding and receptor activation.

Calcitonin (CT) and parathyroid hormone (PTH), whose receptors belong to the same family of G proteincoupled receptors, share no amino acid sequence homology and selectively activate either CT or PTH receptors. We now show, however, that reciprocal hybrid ligands (CT/PTH and PTH/CT), which do not activate the "wildtype" receptors, activate PTH/CT and CT/PTH receptor chimeras, respectively. Our findings indicate that PTH and CT share a similar architecture with at least two functional, receptor-specific domains. These domains are sufficiently independent to permit synthetic hybrid ligands to efficiently activate appropriate receptor chimeras. Therefore, both ligands follow, despite their very different primary sequences, a common pattern of ligand-receptor interaction.The isolation of cDNAs encoding the receptors for secretin (1), calcitonin (CT) 1 (2), and parathyroid hormone (PTH)/PTHrelated peptide (PTHrP) (3, 4) established a new family of G protein-coupled receptors (GPRs) (5, 6). Members of this family, including some invertebrate receptors (7), 2,3 share, in addition to their overall structure with seven membrane-spanning helices, approximately 50 strictly conserved amino acids, including 8 important cysteines, an almost invariant amino acid sequence of the seventh membrane-spanning domain, and a similar intron/exon organization (10 -16). These findings suggest that this family of GPRs may have evolved from a common ancestral precursor. The ligands which activate these GPRs are similar in length, but lack, with the exception of the first 13 residues in PTH and PTHrP, any amino acid sequence homology. For both PTH-(1-34) and CT-(1-32), the importance of the amino terminus for bioactivity has been recognized, whereas the carboxyl-terminal portion contributes predominantly to receptor binding (17)(18)(19)(20)(21)(22)(23). Based on the limited data available, we and others had previously proposed that the carboxyl-terminal portion of these ligands determines specificity for the aminoterminal, extracellular receptor domains, while the amino termini of most ligands functionally interact with the membraneembedded receptor region (23-29). We have now tested this hypothesis more directly by constructing reciprocal chimeric ligands composed of portions of PTH and CT, and reciprocal chimeric receptors composed of portions of the PTH/PTHrP and CT receptor. These chimeras were designed such that the carboxyl-terminal portion of the ligand would match the aminoterminal, extracellular domain of the receptor, while the amino-terminal portion of the hybrid ligand would correspond to the membrane-embedded domains of the receptor and connecting loops. Our functional analysis of these hybrid ligands and chimeric receptors strengthens the proposed model of ligand-receptor interaction. Moreover, our studies imply that the receptors and their ligands are composed of functionally independent domains. ) were synthesized by the MGH Biopolymer Facility using Fmoc (N-(9-fluorenyl)methoxycarbonyl) technology (30). All peptides were H...