Trace amine-associated receptor 1 (TAAR1), a member of the trace amine receptor family, recognizes various trace amines in the brain, including endogenous β-phenylethylamine (PEA) and methamphetamine (METH). TAAR1 is a novel target for several neurological disorders, including schizophrenia, depression, and substance abuse. Herein, we report the structure of the human TAAR1-Gs protein complex bound to METH. Using functional studies, we reveal the molecular basis of METH recognition by TAAR1, and potential mechanisms underlying the selectivity of TAAR1 for different ligands. Molecular dynamics simulations further elucidated possible mechanisms for the binding of chiral amphetamine (AMPH)-like psychoactive drugs to TAAR1. Additionally, we discovered a hydrophobic core on the transmembrane helices (TM), TM5 and TM6, explaining the unique mechanism of TAAR1 activation. These findings reveal the ligand recognition pattern and activation mechanism of TAAR1, which has important implications for the development of next-generation treatments for substance abuse and various neurological disorders.

Hypoparathyroidism, a deficiency of parathyroid hormone (PTH), results in hypocalcemia, hyperphosphatemia, and hypercalciuria. The disease is poorly controlled by calcium and vitamin D supplements or native PTH(1-84) replacement therapy. A version of PTH is being developed using D-VITylation technology, whereby vitamin D is conjugated to a therapeutic peptide, which confers a long plasma half-life by virtue of binding to the abundant vitamin D binding protein (DBP). D-VITylation of PTH caused no reduction in activity at the PTHR1 receptor, and resulted in a plasma elimination half-life of 7-15 h in rats and 24-32 h in cynomolgus monkeys. Analysis of steady-state pharmacokinetics as a function of dose showed flat profiles with smaller peak:trough ratios at low doses, indicative of slower subcutaneous absorption. In thyroparathyroidectomized (TPTx) rats, PTH(1-34)-vitamin D conjugates restored serum calcium and phosphate levels into the normal range over the 24 h dosing period, and increased bone turnover markers and reduced bone mineral density. Urinary calcium was initially elevated, but normalized by the end of treatment on day 27. In healthy monkeys, a single dose of PTH(1-34)-vitamin D conjugates elevated serum calcium levels above the normal range for a period of 24-48 h while simultaneously reducing urinary calcium. Therefore, the lead compound, EXT608, is a promising candidate as a therapeutic that can truly mimic the endogenous activity of PTH and warrants further study in patients with hypoparathyroidism.
© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.

Peptide-receptor interactions play critical roles in a wide variety of physiological processes. Methods to link bioactive peptides covalently to unmodified receptors on the surfaces of living cells are valuable for studying receptor signaling, dynamics, and trafficking and for identifying novel peptide-receptor interactions. Here, we utilize peptide analogues bearing deactivated aryl diazonium groups for the affinity-driven labeling of unmodified receptors. We demonstrate that aryl diazonium-bearing peptide analogues can covalently label receptors on the surface of living cells using both the neurotensin and the glucagon-like peptide 1 receptor systems. Receptor labeling occurs in the complex environment of the cell surface in a sequence-specific manner. We further demonstrate the utility of this covalent labeling approach for the visualization of peptide receptors by confocal fluorescence microscopy and for the enrichment and identification of labeled receptors by mass spectrometry-based proteomics. Aryl diazonium-based affinity-driven receptor labeling is attractive due to the high abundance of tyrosine and histidine residues susceptible to azo coupling in the peptide binding sites of receptors, the ease of incorporation of aryl diazonium groups into peptides, and the relatively small size of the aryl diazonium group. This approach should prove to be a powerful and relatively general method to study peptide-receptor interactions in cellular contexts.

The l- to d-amino acid residue isomerization of neuropeptides is an understudied post-translational modification found in animals across several phyla. Despite its physiological importance, little information is available regarding the impact of endogenous peptide isomerization on receptor recognition and activation. As a result, the full roles peptide isomerization play in biology are not well understood. Here, we identify that the Aplysia allatotropin-related peptide (ATRP) signaling system utilizes l- to d-residue isomerization of one amino acid residue in the neuropeptide ligand to modulate selectivity between two distinct G protein-coupled receptors (GPCRs). We first identified a novel receptor for ATRP that is selective for the D2-ATRP form, which bears a single d-phenylalanine residue at position 2. Using cell-based receptor activation experiments, we then characterized the stereoselectivity of the two known ATRP receptors for both endogenous ATRP diastereomers, as well as for homologous toxin peptides from a carnivorous predator. We found that the ATRP system displayed dual signaling through both the Gαq and Gαs pathways, and each receptor was selectively activated by one naturally occurring ligand diastereomer over the other. Overall, our results provide insights into an unexplored mechanism by which nature regulates intercellular communication. Given the challenges in detecting l- to d-residue isomerization from complex mixtures de novo and in identifying receptors for novel neuropeptides, it is likely that other neuropeptide-receptor systems may also utilize changes in stereochemistry to modulate receptor selectivity in a manner similar to that discovered here.

Agonists selectively targeting cannabinoid receptor-like G-protein-coupled receptor (GPCR) GPR119 hold promise for treating metabolic disorders while avoiding unwanted side effects. Here we present the cryo-electron microscopy (cryo-EM) structures of the human GPR119-Gs signaling complexes bound to AR231453 and MBX-2982, two representative agonists reported for GPR119. The structures reveal a one-amino acid shift of the conserved proline residue of TM5 that forms an outward bulge, opening up a hydrophobic cavity between TM4 and TM5 at the middle of the membrane for its endogenous ligands-monounsaturated lipid metabolites. In addition, we observed a salt bridge between ICL1 of GPR119 and Gβs. Disruption of the salt bridge eliminates the cAMP production of GPR119, indicating an important role of Gβs in GPR119-mediated signaling. Our structures, together with mutagenesis studies, illustrate the conserved binding mode of the chemically different agonists, and provide insights into the conformational changes in receptor activation and G protein coupling.
© 2022. The Author(s).