Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation

G-protein coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signaling effectors such as G proteins and β-arrestins. It is widely appreciated that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (i.e., ‘efficacy’)1. Furthermore, mounting biophysical evidence, primarily using the β-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states2–5. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies): a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60)6,7. We show that Nb60 stabilizes a previously unappreciated low affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoproterenol has a 15,000-fold higher affinity for the β2AR in the presence of Nb80 compared to Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states

Allosteric Nanobodies Reveal the Dynamic Range and Diverse Mechanisms of GPCR Activation

G-protein coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signaling effectors such as G proteins and β-arrestins. It is widely appreciated that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (i.e., ‘efficacy’)1. Furthermore, mounting biophysical evidence, primarily using the β-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states2–5. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies): a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60)6,7. We show that Nb60 stabilizes a previously unappreciated low affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoproterenol has a 15,000-fold higher affinity for the β2AR in the presence of Nb80 compared to Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states

Differential diagnosis of papilledema vs. pseudopapilledema using customized optical coherence tomography parameters

Objective measures of the optic nerve head (ONH) and peripapillary tissue with optical coherence tomography (OCT) are useful for diagnosis of papilledema (PE) but limited to retinal nerve fiber layer (RNFL) thickness from a circular scan path centered on the ONH. The purpose of this study was to evaluate the use of additional OCT-derived measures in differentiating PE from pseudopapilledema (PPE

Effects of topical insulin administration on phosphorylated protein kinase B and nitric oxide levels in the rat retina

Purpose: To examine the effects of insulin treatment on the expression of phosphorylated protein kinase B (Akt or pAkt) and nitric oxide (NO), known targets of insulin action. Methods: Retinas from Lewis rats were treated with insulin in vitro, and the levels of pAkt and NO expression assessed in lysed tissue homogenates at various time points. In addition, rats were treated in vivo with a single eye drop containing 1% insulin after which the levels of pAkt and NO were assessed in their retinas at various time points. Results: Incubation of retinas in media containing 10 or 100 ng/mL of insulin resulted in a significant increase in their expression of pAkt 15–30 min later. When insulin was topically applied in vivo, its levels peaked in the retina 35 min. Topically applied insulin significantly increased retinal pAkt levels 10 and 15 min post-treatment (p < 0.02 and 0.01, respectively). NO levels were elevated 20 min after insulin treatment both in vitro and in vivo (p < 0.007 and 0.04, respectively). Conclusions: Insulin upregulated retinal expression of pAkt and NO both in vivo and in vitro, suggesting that topically applied insulin that accumulates in the retina may be physiologically active

Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies

The biologic activity induced by ligand binding to orthosteric or allosteric sites on a G protein–coupled receptor (GPCR) is mediated by stabilization of specific receptor conformations. In the case of the β(2) adrenergic receptor, these ligands are generally small-molecule agonists or antagonists. However, a monomeric single-domain antibody (nanobody) from the Camelid family was recently found to allosterically bind and stabilize an active conformation of the β(2)-adrenergic receptor (β(2)AR). Here, we set out to study the functional interaction of 18 related nanobodies with the β(2)AR to investigate their roles as novel tools for studying GPCR biology. Our studies revealed several sequence-related nanobody families with preferences for active (agonist-occupied) or inactive (antagonist-occupied) receptors. Flow cytometry analysis indicates that all nanobodies bind to epitopes displayed on the intracellular receptor surface; therefore, we transiently expressed them intracellularly as “intrabodies” to test their effects on β(2)AR-dependent signaling. Conformational specificity was preserved after intrabody conversion as demonstrated by the ability for the intracellularly expressed nanobodies to selectively bind agonist- or antagonist-occupied receptors. When expressed as intrabodies, they inhibited G protein activation (cyclic AMP accumulation), G protein–coupled receptor kinase (GRK)–mediated receptor phosphorylation, β-arrestin recruitment, and receptor internalization to varying extents. These functional effects were likely due to either steric blockade of downstream effector (G(s), β-arrestin, GRK) interactions or stabilization of specific receptor conformations which do not support effector coupling. Together, these findings strongly implicate nanobody-derived intrabodies as novel tools to study GPCR biology

Changes in Optic Nerve Head and Retinal Morphology During Spaceflight and Acute Fluid Shift Reversal

Importance: Countermeasures that reverse the headward fluid shift experienced in weightlessness have the potential to mitigate spaceflight-associated neuro-ocular syndrome. This study investigated whether use of the countermeasure lower-body negative pressure during spaceflight was associated with changes in ocular structure. Objective: To determine whether changes to the optic nerve head and retina during spaceflight can be mitigated by brief in-flight application of 25-mm Hg lower-body negative pressure. Design, Setting, and Participants: In the National Aeronautics and Space Administration\u27s Fluid Shifts Study, a prospective cohort study, optical coherence tomography scans of the optic nerve head and macula were obtained from US and international crew members before flight, in-flight, and up to 180 days after return to Earth. In-flight scans were obtained both under normal weightless conditions and 10 to 20 minutes into lower-body negative pressure exposure. Preflight and postflight data were collected in the seated, supine, and head-down tilt postures. Crew members completed 6- to 12-month missions that took place on the International Space Station. Data were analyzed from 2016 to 2021. Interventions or Exposures: Spaceflight and lower-body negative pressure. Main Outcomes and Measures: Changes in minimum rim width, optic cup volume, Bruch membrane opening height, peripapillary total retinal thickness, and macular thickness. Results: Mean (SD) flight duration for the 14 crew members (mean [SD] age, 45 [6] years; 11 male crew members [79%]) was 214 (72) days. Ocular changes on flight day 150, as compared with preflight seated, included an increase in minimum rim width (33.8 μm; 95% CI, 27.9-39.7 μm; P \u3c .001), decrease in cup volume (0.038 mm3; 95% CI, 0.030-0.046 mm3; P \u3c .001), posterior displacement of Bruch membrane opening (-9.0 μm; 95% CI, -15.7 to -2.2 μm; P = .009), and decrease in macular thickness (fovea to 500 μm, 5.1 μm; 95% CI, 3.5-6.8 μm; P \u3c .001). Brief exposure to lower-body negative pressure did not affect these parameters. Conclusions and Relevance: Results of this cohort study suggest that peripapillary tissue thickening, decreased cup volume, and mild central macular thinning were associated with long-duration spaceflight. Acute exposure to 25-mm Hg lower-body negative pressure did not alter optic nerve head or retinal morphology, suggesting that longer durations of a fluid shift reversal may be needed to mitigate spaceflight-induced changes and/or other factors are involved

Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation.

G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and β-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’). Furthermore, increasing biophysical evidence, primarily using the β2-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for β2AR in the presence of Nb80 compared to the affinity of isoprenaline for β2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states