Molecular insights into differentiated ligand recognition of the human parathyroid hormone receptor 2

The parathyroid hormone receptor 2 (PTH2R) is a class B1 G protein–coupled receptor (GPCR) involved in the regulation of calcium transport, nociception mediation, and wound healing. Naturally occurring mutations in PTH2R were reported to cause hereditary diseases, including syndromic short stature. Here, we report the cryogenic electron microscopy structure of PTH2R bound to its endogenous ligand, tuberoinfundibular peptide (TIP39), and a heterotrimeric G_s protein at a global resolution of 2.8 Å. The structure reveals that TIP39 adopts a unique loop conformation at the N terminus and deeply inserts into the orthosteric ligand-binding pocket in the transmembrane domain. Molecular dynamics simulation and site-directed mutagenesis studies uncover the basis of ligand specificity relative to three PTH2R agonists, TIP39, PTH, and PTH-related peptide. We also compare the action of TIP39 with an antagonist lacking six residues from the peptide N terminus, TIP(7-39), which underscores the indispensable role of the N terminus of TIP39 in PTH2R activation. Additionally, we unveil that a disease-associated mutation G258D significantly diminished cAMP accumulation induced by TIP39. Together, these results not only provide structural insights into ligand specificity and receptor activation of class B1 GPCRs but also offer a foundation to systematically rationalize the available pharmacological data to develop therapies for various disorders associated with PTH2R.

Class B1 G protein–coupled receptors (GPCRs) comprise 15 members involved in a wide spectrum of physiological functions (1, 2). A number of them are validated drug targets for different human diseases, such as osteoporosis, type 2 diabetes, obesity, psychiatric disorders, and migraine. Among them are two types of parathyroid hormone (PTH) receptors (PTH1R and PTH2R), whose actions are mediated by coupling primarily to the stimulatory G protein (G_s) (3, 4). Expressed in the central and peripheral nervous systems, PTH2R is a key mediator of nociception, wound healing, and maternal behavior (5–8). Furthermore, recent studies have shown that it regulates calcium transport and influences keratinocyte differentiation, pointing to its potential in the treatment of Darier disease or Hailey–Hailey disease (9). Furthermore, naturally occurring PTH2R mutations have been linked to familial early-onset generalized osteoarthritis, syndromic intellectual disability, and syndromic short stature (10, 11). The latter is presently being treated with recombinant human growth hormone (12).PTH receptors have three endogenous ligands, namely, tuberoinfundibular peptide of 39 residues (TIP39), PTH, and PTH-related peptide (PTHrP). Unlike PTH and PTHrP that mainly expressed in peripheral systems, TIP39-containing neuronal cell bodies have been identified in the subparafascicular area and the medial paralemniscal nucleus (13). Both PTH and PTHrP are implicated in skeletal development, calcium homeostasis, and bone turnover (14). In fact, PTH (1-34) and abaloparatide, a variant of PTHrP (1-34) (15), are Food and Drug Administration–approved drugs for osteoporosis. Discovered in the bovine hypothalamus, TIP39 contains two identical and several similar residues common to PTH and PTHrP. However, there is no evidence to suggest that TIP39 plays a role in mineral or bone metabolism. In contrast to PTH that indistinguishably activates both receptors, TIP39 is selective for PTH2R (13, 16), while PTHrP only has a weak action on PTH2R (3, 4, 13). Deletion of six residues from the N terminus of TIP39 results in a PTH2R antagonist, TIP(7-39) (17). However, the underlying mechanism by which PTH2R selectively recognizes these related but distinct peptides is largely unknown. Although newly solved cryogenic electron microscopy (cryo-EM) structure of LA-PTH–PTH1R–G_s complex offers valuable insights into PTH recognition and receptor activation (18), questions remain relative to their applicability to PTH2R. Thus, we determined the single-particle cryo-EM structure of the human PTH2R in complex with TIP39 and a heterotrimeric G_s protein at a global resolution of 2.8 Å. Together with molecular dynamics (MD) simulation results, it provides an in-depth understanding of the structural basis of ligand specificity and PTH2R activation.