Structural insights into differences in G protein activation by family A and family B GPCRs.

Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) play important roles in carbohydrate metabolism. Recent structures of family B GPCR-Gs protein complexes reveal a disruption in the α-helix of transmembrane segment 6 (TM6) not observed in family A GPCRs. To investigate the functional impact of this structural difference, we compared the structure and function of the glucagon receptor (GCGR; family B) with the ß2 adrenergic receptor (ß2AR; family A). We determined the structure of the GCGR-Gs complex by means of cryo-electron microscopy at 3.1-angstrom resolution. This structure shows the distinct break in TM6. Guanosine triphosphate (GTP) turnover, guanosine diphosphate release, GTP binding, and G protein dissociation studies revealed much slower rates for G protein activation by the GCGR compared with the ß2AR. Fluorescence and double electron-electron resonance studies suggest that this difference is due to the inability of agonist alone to induce a detectable outward movement of the cytoplasmic end of TM6.

Biased agonism of clinically approved µ-opioid receptor agonists and TRV130 is not controlled by binding and signaling kinetics.

Binding and signaling kinetics have previously proven important in validation of biased agonism at GPCRs. Here we provide a comprehensive kinetic pharmacological comparison of clinically relevant µ-opioid receptor agonists, including the novel biased agonist oliceridine (TRV130) which is in clinical trial for pain management. We demonstrate that the bias profile observed for the selected agonists is not time-dependent and that agonists with dramatic differences in their binding kinetic properties can display the same degree of bias. Binding kinetics analyses demonstrate that buprenorphine has 18-fold higher receptor residence time than oliceridine. This is thus the largest pharmacodynamic difference between the clinically approved drug buprenorphine and the clinical candidate oliceridine, since their bias profiles are similar. Further, we provide the first pharmacological characterization of (S)-TRV130 demonstrating that it has a similar pharmacological profile as the (R)-form, oliceridine, but displays 90-fold lower potency than the (R)-form. This difference is driven by a significantly slower association rate. Finally, we show that the selected agonists are differentially affected by G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) expression. GRK2 and GRK5 overexpression greatly increased µ-opioid receptor internalization induced by morphine, but only had modest effects on buprenorphine and oliceridine-induced internalization. Overall, our data reveal that the clinically available drug buprenorphine displays a similar pharmacological bias profile in vitro compared to the clinical candidate drug oliceridine and that this bias is independent of binding kinetics suggesting a mechanism driven by receptor-conformations. This article is part of the Special Issue entitled 'New Vistas in Opioid Pharmacology'.

Human GIP(3-30)NH2 inhibits G protein-dependent as well as G protein-independent signaling and is selective for the GIP receptor with high-affinity binding to primate but not rodent GIP receptors.

GIP(3-30)NH2 is a high affinity antagonist of the GIP receptor (GIPR) in humans inhibiting insulin secretion via G protein-dependent pathways. However, its ability to inhibit G protein-independent signaling is unknown. Here we determine its action on arrestin-recruitment and receptor internalization in recombinant cells. As GIP is adipogenic, we evaluate the inhibitory actions of GIP(3-30)NH2 in human adipocytes. Finally, we determine the receptor selectivity of GIP(3-30)NH2 among other human and animal GPCRs. cAMP accumulation and ß-arrestin 1 and 2 recruitment were studied in transiently transfected HEK293 cells and real-time internalization in transiently transfected HEK293A and in HEK293A ß-arrestin 1 and 2 knockout cells. Furthermore, human subcutaneous adipocytes were assessed for cAMP accumulation following ligand stimulation. Competition binding was examined in transiently transfected COS-7 cells using human 125I-GIP(3-30)NH2. The selectivity of human GIP(3-30)NH2 was examined by testing for agonistic and antagonistic properties on 62 human GPCRs. Human GIP(3-30)NH2 inhibited GIP(1-42)-induced cAMP and ß-arrestin 1 and 2 recruitment on the human GIPR and Schild plot analysis showed competitive antagonism with a pA2 and Hill slope of 16.8 nM and 1.11 ±â€¯0.02 in cAMP, 10.6 nM and 1.15 ±â€¯0.05 in ß-arrestin 1 recruitment, and 10.2 nM and 1.06 ±â€¯0.05 in ß-arrestin 2 recruitment. Efficient internalization of the GIPR was dependent on the presence of either ß-arrestin 1 or 2. Moreover, GIP(3-30)NH2 inhibited GIP(1-42)-induced internalization in a concentration-dependent manner and notably also inhibited GIP-mediated signaling in human subcutaneous adipocytes. Finally, the antagonist was established as GIPR selective among 62 human GPCRs being species-specific with high affinity binding to the human and non-human primate (Macaca fascicularis) GIPRs, and low affinity binding to the rat and mouse GIPRs (Kd values of 2.0, 2.5, 31.6 and 100 nM, respectively). In conclusion, human GIP(3-30)NH2 is a selective and species-specific GIPR antagonist with broad inhibition of signaling and internalization in transfected cells as well as in human adipocytes.