PTH/PTHrP Receptor Signaling, Allostery, and Structures

Abstract

The parathyroid hormone (PTH) type 1 receptor (PTHR) is the canonical G protein-coupled receptor (GPCR) for PTH and PTH-related protein (PTHrP) and the key regulator of calcium homeostasis and bone turnover. PTHR function is critical for human health to maintain homeostatic control of ionized serum Ca²⁺ levels and has several unusual signaling features, such as endosomal cAMP signaling, that are well-studied but not structurally understood. In this review, we discuss how recently solved high resolution near-atomic structures of hormone-bound PTHR in its inactive and active signaling states and discovery of extracellular Ca²⁺ allosterism shed light on the structural basis for PTHR signaling and function.

Keywords: G protein-coupled receptor; allostery; cAMP; cryo-EM structure; endosomes; parathyroid hormone.

Figure 1.. Modes of Parathyroid Hormone Type 1 Receptor (PTHR) Signaling.

(A) Major G protein signaling pathways triggered by PTHR activation. General principle of signaling by PTHR. Parathyroid hormone (PTH) or PTH-related protein (PTHrP) binding induces or stabilizes active PTHR conformations, which promote coupling and activation of heterotrimeric G proteins. G_s activates adenylate cyclases (AC), leading to cAMP synthesis and activation of protein kinase A (PKA). Gq activates phospholipase C, which cleaves phosphatidylinositol (4,5)-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol (1,4,5)-trisphosphate (IP3). IP3 then diffuses through the cytosol and activates IP3-gated Ca²⁺ channels located in the endoplasmic reticulum membrane, releasing stored Ca²⁺ into the cytosol. An increase in cytosolic Ca²⁺ promotes PKC translocation to the plasma membrane and then activation by DAG. (B) An additional mode of cAMP signaling has been revealed, where internalized PTHR in endosomes also prolongs cAMP production, which diffuses to the nucleus to directly activate nuclear PKA (blue box). Details are discussed in Box 2.

Figure 2.. Cryo-Electron Microscopy Structure of Active State Parathyroid Hormone Type 1 Receptor (PTHR) Bound to Long Acting Parathyroid Hormone (LA-PTH) and in Complex with Gs Heterotrimer.

(A) Overall structure of the complex in cartoon and transparent surface representation; PTHR, LA-PTH, Gαs, Gβ, and Gγ are shown in violet, orange, green, cyan, and magenta, respectively (same color scheme in next panels). Light orange rectangle represents the putative membrane bilayer boundaries. (B) Close-up view of Gs heterotrimer–PTHR interaction interface. The interacting residues are shown as sticks and contacts are represented by orange broken lines (polar interactions ≤4 Å, hydrophobic interactions <5 Å). (C) Sites of PTHR mutations causing Jansen’s metaphyseal chondroplasia (JMC). Wild type residues H223, T410, and I458 are shown as yellow sticks. Single point mutation to R223, P/R410, or R/K458 causes the disease. (D) Alignment of Gαs structures. The receptor-bound state is state 1 of the LA-PTH–PTHR–Gs structure. The receptor-free state is the crystal structure of GTPγs-bound Gαs (Protein Data Bank 1AZT), colored pink. GTPγs is shown as cyan sticks. The GDP/GTP binding site of Gαs is circled blue. (E) Hydrophobic interactions between F376 (yellow) and other Gαs residues, shown as sticks, in the receptor-bound form. (F) Hydrophobic interactions between F376 (magenta) and other Gαs residues, shown as sticks, in the receptor-free form. Abbreviation: TMD, transmembrane domain.

Figure 3.. Structural Dynamics of Long Acting Parathyroid Hormone (LA-PTH)–Parathyroid Hormone Type 1 Receptor (PTHR) Complex.

(A) Three distinct conformational states of LA-PTH–PTHR complex obtained from cryo-electron microscopy data analysis. PTHR and LA-PTH are shown as different shades of violet and orange, respectively. The oscillation of the N-terminal domain of PTHR (N-PTHR) is evident, while the transmembrane domain (TMD) core shows higher degree of stability, likely due to tight binding of ^NLA-PTH. Notably, state 3 captures the event of partial dissociation of ^CLA-PTH from N-PTHR (yellow helix). (B) Potential mechanism of ^CLA-PTH dissociation, with LA-PTH D17 and PTHR D137 and E177 shown as sticks. Left, states 1 and 2. Right, state 3.

Figure 4.. Structures of Parathyroid Hormone Type 1 Receptor (PTHR) in Active (A–C) and Inactive (D–F) States.

(A) Residue interaction network within the receptor transmembrane domain (TMD) core of the active structure. Interacting residues (with interaction distances ≤4 Å) are shown as sticks and contacts are represented by cyan broken lines. Long acting parathyroid hormone (LA-PTH) and TM6 of the receptor are shown in orange and violet, respectively. TM1 and TM7 are transparent cartoons for clarity. (B) Close-up view demonstrating the key activating polar interactions of E4 of LA-PTH with Y195 and R233 of the receptor. (C) Close-up top view showing the position and interaction of the N-terminal tip of LA-PTH with receptor TM6. (D) Amino acid interaction network within the receptor TMD core of the inactive structure (pdb 6fj3). Interacting residues (with interaction distances ≤4 Å) are shown as sticks and contacts are represented by cyan broken lines. LA-PTH and TM6 of the receptor are shown in pink and violet, respectively. TM1 and TM7 are transparent cartoons for clarity. (E) Close-up view demonstrating the key activating polar interactions of E4 of ePTH with Y195 and R233 of the receptor. The failure to switch the receptor to its active state is likely due to multiple thermostabilizing mutations in the receptor. E4 makes an additional contact with N448 in the ePTH–PTHR structure. (F) Close-up top view showing the position and interaction of the N-terminal tip of ePTH with the TM5 of the receptor. (G) Sequence alignment of PTH, ePTH, and LA-PTH. Residues interacting with the receptor TMD are highlighted in lilac. Unnatural amino acids of ePTH: A*, aminocyclopentane-1-carboxylic acid; A^§, α-aminoisobutyric acid; R^§, homoarginine; Lⁿ, norleucine.

Figure 5.. Overlay of Inactive and Active Parathyroid Hormone Type 1 Receptor (PTHR) Structures (A,B) and Structural Basis of Parathyroid Hormone (PTH)-R25 Contribution to Ca²⁺ Allostery (C–E).

The receptor and modified PTH (ePTH) of the inactive structure are colored dark red and pink, respectively. The receptor and long acting parathyroid hormone (LA-PTH) of the active structure are colored violet and orange, respectively. (A) Alignment of the two structures by transmembrane domain (TMD) residues 180–460. (B) Structural alignment by the N-terminal domain of PTHR (N-PTHR) residues (27–168), showcasing similar N-PTHR conformations between the two structures. (C) Active PTHR state 1, with LA-PTH H25 and PTHR L174 shown as sticks. The receptor and LA-PTH of the active structure are colored violet and orange, respectively. Extracellular loop 1 (ECL1) start/end points indicated by a broken box. (D) Inactive PTHR structure, with ePTH R25 and PTHR L174 shown as sticks. The receptor and ePTH of the inactive structure are colored dark red and pink, respectively. ECL1 location indicated by a broken box. (E) Schematic diagram of Ca²⁺ coordination by PTH-PTHR. PTH R25 interactions with two acidic clusters in ECL1, permitting these clusters to coordinate Ca²⁺.