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The human histamine H3 receptor (hH3R) is predominantly expressed in the CNS, where it regulates the synthesis and release of histamine and other neurotransmitters. Due to its neuromodulatory role, the hH3R has been associated with various CNS disorders, including Alzheimer’s and Parkinson’s disease. Markedly, the hH3R gene undergoes extensive splicing, resulting in 20 isoforms, of which 7TM isoforms exhibit variations in the intracellular loop 3 (IL3) and/or C-terminal tail. Particularly, hH3R isoforms that display variations in IL3 (e.g., hH3R-365) are shown to differentially signal via Gαi-dependent pathways upon binding of biased agonists (e.g., immepip, proxifan, imetit). Nevertheless, the mechanisms underlying biased agonism at hH3R isoforms remain unknown. Using a structure-function relationship study with a broad range of H3R agonists, we thereby explored determinants underlying isoform bias at hH3R isoforms that exhibit variations in IL3 (i.e., hH3R-445, -415, -365, and -329) in a Gαi-dependent pathway (cAMP inhibition). Hence, we systematically characterized hH3R isoforms on isoform bias by comparing various ligand properties (i.e., structural and molecular) to the degree of isoform bias. Importantly, our study provides novel insights into the structural and molecular basis of receptor isoform bias, highlighting the importance to study GPCRs with multiple isoforms to better tailor drugs.

The calcium-sensing receptor (CaSR), a cell-surface sensor for Ca2+, is the master regulator of calcium homeostasis in humans and is the target of calcimimetic drugs for the treatment of parathyroid disorders1. CaSR is a family C G-protein-coupled receptor2 that functions as an obligate homodimer, with each protomer composed of a Ca2+-binding extracellular domain and a seven-transmembrane-helix domain (7TM) that activates heterotrimeric G proteins. Here we present cryo-electron microscopy structures of near-full-length human CaSR in inactive or active states bound to Ca2+ and various calcilytic or calcimimetic drug molecules. We show that, upon activation, the CaSR homodimer adopts an asymmetric 7TM configuration that primes one protomer for G-protein coupling. This asymmetry is stabilized by 7TM-targeting calcimimetic drugs adopting distinctly different poses in the two protomers, whereas the binding of a calcilytic drug locks CaSR 7TMs in an inactive symmetric configuration. These results provide a detailed structural framework for CaSR activation and the rational design of therapeutics targeting this receptor. Cryo-EM structures of human calcium-sensing receptor reveal intrinsic asymmetry in the receptor homodimer upon activation that is stabilized by calcimimetic drugs adopting distinct poses in the two protomers, priming one protomer for G-protein coupling.

Real-time label-free techniques are used to profile G protein-coupled receptor (GPCR) signaling pathways in living cells. However, interpreting the label-free signal responses is challenging, and previously reported methods do not reliably separate pathways from each other. In this study, a continuous angular-scanning surface plasmon resonance (SPR) technique is utilized for measuring label-free GPCR signal profiles. We show how the continuous angular-scanning ability, measuring up to nine real-time label-free parameters simultaneously, results in more information-rich label-free signal profiles for different GPCR pathways, providing a more accurate pathway separation. For this, we measured real-time full-angular SPR response curves for Gs, Gq, and Gi signaling pathways in living cells. By selecting two of the most prominent label-free parameters: the full SPR curve angular and intensity shifts, we present how this analysis approach can separate each of the three signaling pathways in a straightforward single-step analysis setup, without concurrent use of signal inhibitors or other response modulating compounds.