Development and Characterization of Pepducins as Gs-biased Allosteric Agonists

The β₂-adrenergic receptor (β₂AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. β-Agonists used to combat hypercontractility in airway smooth muscle stimulate β₂AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the β₂AR by long acting β-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. β₂AR desensitization to β-agonists is primarily mediated by G protein-coupled receptor kinases and β-arrestins that attenuate receptor-G_s coupling and promote β₂AR internalization and degradation. A biased agonist that can selectively stimulate G_s signaling without promoting receptor interaction with G protein-coupled receptor kinases and β-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened ∼50 lipidated peptides derived from the intracellular loops of the β₂AR, known as pepducins. This screen revealed two classes of G_s-biased pepducins, receptor-independent and receptor-dependent, as well as several β-arrestin-biased pepducins. The receptor-independent G_s-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent G_s-biased pepducins appear to stabilize a G_s-biased conformation of the β₂AR that couples to G_s but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or β-arrestin-mediated internalization. Functional studies in primary human airway smooth muscle cells demonstrate that G_s-biased pepducins are not subject to conventional desensitization and thus may be good candidates for the development of next generation asthma therapeutics. Our study reports the first G_s-biased activator of the β₂AR and provides valuable tools for the study of β₂AR function.     

Monoclonal anti-β1-adrenergic receptor antibodies activate G protein signaling in the absence of β-arrestin recruitment

Thermostabilized G protein-coupled receptors used as antigens for in vivo immunization have resulted in the generation of functional agonistic anti-β₁-adrenergic (β₁AR) receptor monoclonal antibodies (mAbs). The focus of this study was to examine the pharmacology of these antibodies to evaluate their mechanistic activity at β₁AR. Immunization with the β₁AR stabilized receptor yielded five stable hybridoma clones, four of which expressed functional IgG, as determined in cell-based assays used to evaluate cAMP stimulation. The antibodies bind diverse epitopes associated with low nanomolar agonist activity at β₁AR, and they appeared to show some degree of biased signaling as they were inactive in an assay measuring signaling through β-arrestin. In vitro characterization also verified different antibody-receptor interactions reflecting the different epitopes on the extracellular surface of β₁AR to which the mAbs bind. The anti-β₁AR mAbs only demonstrated agonist activity when in dimeric antibody format, but not as the monomeric Fab format, suggesting that agonist activation may be mediated through promoting receptor dimerization. Finally, we have also shown that at least one of these antibodies exhibits in vivo functional activity at a therapeutically-relevant dose producing an increase in heart rate consistent with β₁AR agonism.     

Two Classes of Cholesterol Binding Sites for the β2AR Revealed by?Thermostability and NMR

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the β₂ adrenergic receptor (β₂AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β₂AR. By analyzing the β₂AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β₂AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.     

Molecular Basis of Cannabinoid CB1 Receptor Coupling to the G Protein Heterotrimer Gαiβγ: IDENTIFICATION OF KEY CB1 CONTACTS WITH THE C-TERMINAL HELIX α5 OF Gαi*

The cannabinoid (CB1) receptor is a member of the rhodopsin-like G protein-coupled receptor superfamily. The human CB1 receptor, which is among the most expressed receptors in the brain, has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. Different classes of CB1 agonists evoke signaling pathways through the activation of specific subtypes of G proteins. The molecular basis of CB1 receptor coupling to its cognate G protein is unknown. As a first step toward understanding CB1 receptor-mediated G protein signaling, we have constructed a ternary complex structural model of the CB1 receptor and G_i heterotrimer (CB1-G_i), guided by the x-ray structure of β₂-adrenergic receptor (β₂AR) in complex with G_s (β₂AR-G_s), through 824-ns duration molecular dynamics simulations in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer environment. We identified a group of residues at the juxtamembrane regions of the intracellular loops 2 and 3 (IC2 and IC3) of the CB1 receptor, including Ile-218^3.54, Tyr-224^IC2, Asp-338^6.30, Arg-340^6.32, Leu-341^6.33, and Thr-344^6.36, as potential key contacts with the extreme C-terminal helix α₅ of Gα_i. Ala mutations of these residues at the receptor-G_i interface resulted in little G protein coupling activity, consistent with the present model of the CB1-G_i complex, which suggests tight interactions between CB1 and the extreme C-terminal helix α₅ of Gα_i. The model also suggests that unique conformational changes in the extreme C-terminal helix α₅ of Gα play a crucial role in the receptor-mediated G protein activation.     

Assembly of a Ca2+-dependent BK channel signaling complex by binding to beta2 adrenergic receptor

Large-conductance voltage and Ca²⁺-activated potassium channels (BKCa) play a critical role in modulating contractile tone of smooth muscle, and neuronal processes. In most mammalian tissues, activation of β-adrenergic receptors and protein kinase A (PKA_c) increases BKCa channel activity, contributing to sympathetic nervous system/hormonal regulation of membrane excitability. Here we report the requirement of an association of the β2-adrenergic receptor (β2AR) with the pore forming α subunit of BKCa and an A-kinase-anchoring protein (AKAP79/150) for β2 agonist regulation. β2AR can simultaneously interact with both BKCa and L-type Ca²⁺ channels (Ca_v1.2) in vivo, which enables the assembly of a unique, highly localized signal transduction complex to mediate Ca²⁺- and phosphorylation-dependent modulation of BKCa current. Our findings reveal a novel function for G protein-coupled receptors as a scaffold to couple two families of ion channels into a physical and functional signaling complex to modulate β-adrenergic regulation of membrane excitability.     

Identification of GPCR-Interacting Cytosolic Proteins Using HDL Particles and Mass Spectrometry-Based Proteomic Approach

G protein-coupled receptors (GPCRs) have critical roles in various physiological and pathophysiological processes, and more than 40% of marketed drugs target GPCRs. Although the canonical downstream target of an agonist-activated GPCR is a G protein heterotrimer; there is a growing body of evidence suggesting that other signaling molecules interact, directly or indirectly, with GPCRs. However, due to the low abundance in the intact cell system and poor solubility of GPCRs, identification of these GPCR-interacting molecules remains challenging. Here, we establish a strategy to overcome these difficulties by using high-density lipoprotein (HDL) particles. We used the β₂-adrenergic receptor (β₂AR), a GPCR involved in regulating cardiovascular physiology, as a model system. We reconstituted purified β₂AR in HDL particles, to mimic the plasma membrane environment, and used the reconstituted receptor as bait to pull-down binding partners from rat heart cytosol. A total of 293 proteins were identified in the full agonist-activated β₂AR pull-down, 242 proteins in the inverse agonist-activated β₂AR pull-down, and 210 proteins were commonly identified in both pull-downs. A small subset of the β₂AR-interacting proteins isolated was confirmed by Western blot; three known β₂AR-interacting proteins (Gsα, NHERF-2, and Grb2) and 3 newly identified known β₂AR-interacting proteins (AMPKα, acetyl-CoA carboxylase, and UBC-13). Profiling of the identified proteins showed a clear bias toward intracellular signal transduction pathways, which is consistent with the role of β₂AR as a cell signaling molecule. This study suggests that HDL particle-reconstituted GPCRs can provide an effective platform method for the identification of GPCR binding partners coupled with a mass spectrometry-based proteomic analysis.     

A Long Lasting β1 Adrenergic Receptor Stimulation of cAMP/Protein Kinase A (PKA) Signal in Cardiac Myocytes

Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β₁ adrenergic receptor (β₁AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t_½ of 3.9 h) in cardiac myocytes. However, the β₁AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β₁AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β₁AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.     

Differential Signaling of the Endogenous Agonists at the β2-Adrenergic Receptor

The concept of “functional selectivity” or “biased signaling” suggests that a ligand can have distinct efficacies with regard to different signaling pathways. We have investigated the question of whether biased signaling may be related to distinct agonist-induced conformational changes in receptors using the β₂-adrenergic receptor (β₂AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged β₂AR FRET sensor. In this β₂AR sensor, norepinephrine caused signals that amounted to only ≈50% of those induced by epinephrine and the standard “full” agonist isoproterenol. Furthermore, norepinephrine-induced changes in the β₂AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial β₂AR activation signal was revealed for the synthetic agonists fenoterol and terbutaline. However, norepinephrine was almost as efficient as epinephrine (and isoproterenol) in causing activation of G_s and adenylyl cyclase. In contrast, fenoterol was quite efficient in triggering β-arrestin2 recruitment to the cell surface and its interaction with β₂AR, as well as internalization of the receptors, whereas norepinephrine caused partial and slow changes in these assays. We conclude that partial agonism of norepinephrine at the β₂AR is related to the induction of a different active conformation and that this conformation is efficient in signaling to G_s and less efficient in signaling to β-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the β₂AR and link it to distinct conformational changes in the receptor.     

Computational Study on the Different Ligands Induced Conformation Change of β2 Adrenergic Receptor-Gs Protein Complex

β₂ adrenergic receptor (β₂AR) regulated many key physiological processes by activation of a heterotrimeric GTP binding protein (Gs protein). This process could be modulated by different types of ligands. But the details about this modulation process were still not depicted. Here, we performed molecular dynamics (MD) simulations on the structures of β₂AR-Gs protein in complex with different types of ligands. The simulation results demonstrated that the agonist BI-167107 could form hydrogen bonds with Ser203^5.42, Ser207^5.46 and Asn293^6.55 more than the inverse agonist ICI 118,551. The different binding modes of ligands further affected the conformation of β₂AR. The energy landscape profiled the energy contour map of the stable and dissociated conformation of Gαs and Gβγ when different types of ligands bound to β₂AR. It also showed the minimum energy pathway about the conformational change of Gαs and Gβγ along the reaction coordinates. By using interactive essential dynamics analysis, we found that Gαs and Gβγ domain of Gs protein had the tendency to separate when the inverse agonist ICI 118,551 bound to β₂AR. The α5-helix had a relatively quick movement with respect to transmembrane segments of β₂AR when the inverse agonist ICI 118,551 bound to β₂AR. Besides, the analysis of the centroid distance of Gαs and Gβγ showed that the Gαs was separated from Gβγ during the MD simulations. Our results not only could provide details about the different types of ligands that induced conformational change of β₂AR and Gs protein, but also supplied more information for different efficacies of drug design of β₂AR.     

Resolving Two-dimensional Kinetics of the Integrin αIIbβ3-Fibrinogen Interactions Using Binding-Unbinding Correlation Spectroscopy

Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbβ3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbβ3 molecules and forced dissociation of an αIIbβ3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbβ3 pre-exists in at least two states that differ in their zero force on-rates (k_on1 = 1.4 × 10⁻⁴ and k_on2 = 2.3 × 10⁻⁴ μm²/s), off-rates (k_off1 = 2.42 and k_off2 = 0.60 s⁻¹), and dissociation constants (K_d1 = 1.7 × 10⁴ and K_d2 = 2.6 × 10³ μm⁻²). The integrin activator Mn²⁺ changed the on-rates and affinities (K_d1 = 5 × 10⁴ and K_d2 = 0.3 × 10³ μm⁻²) but did not affect the off-rates. The strength of αIIbβ3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbβ3-fibrinogen complex characterized by a faster on-rate. Upon Mn²⁺-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbβ3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbβ3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.     

Structural Features of β2 Adrenergic Receptor: Crystal Structures and Beyond

The beta2-adrenergic receptor (β2AR) family, which is the largest family of cell surface receptors in humans. Extra attention has been focused on the human GPCRs because they have been studied as important protein targets for pharmaceutical drug development. In fact, approximately 40% of marketed drugs directly work on GPCRs. GPCRs respond to various extracellular stimuli, such as sensory signals, neurotransmitters, chemokines, and hormones, to induce structural changes at the cytoplasmic surface, activating downstream signaling pathways, primarily through interactions with heterotrimeric G proteins or through G-protein independent pathways, such as arrestin. Most GPCRs, except for rhodhopsin, which contains covalently linked 11 cis-retinal, bind to diffusible ligands, having various conformational states between inactive and active structures. The first human GPCR structure was determined using an inverse agonist bound β2AR in 2007 and since then, more than 20 distinct GPCR structures have been solved. However, most GPCR structures were solved as inactive forms, and an agonist bound fully active structure is still hard to obtain. In a structural point of view, β2AR is relatively well studied since its fully active structure as a complex with G protein as well as several inactive structures are available. The structural comparison of inactive and active states gives an important clue in understanding the activation mechanism of β2AR. In this review, structural features of inactive and active states of β2AR, the interaction of β2AR with heterotrimeric G protein, and the comparison with β1AR will be discussed.     

Cytoskeletal Reorganization by G Protein-Coupled Receptors Is Dependent on Phosphoinositide 3-Kinase γ, a Rac Guanosine Exchange Factor, and Rac

Reorganization of the actin cytoskeleton is an early cellular response to a variety of extracellular signals. Dissection of pathways leading to actin rearrangement has focused largely on those initiated by growth factor receptors or integrins, although stimulation of G protein-coupled receptors also leads to cytoskeletal changes. In transfected Cos-7SH cells, activation of the chemoattractant formyl peptide receptor induces cortical actin polymerization and a decrease in the number of central actin bundles. In this report, we show that cytoskeletal reorganization can be transduced by G protein βγ heterodimers (G_βγ), phosphoinositide 3-kinase γ (PI3-K_γ), a guanosine exchange factor (GEF) for Rac, and Rac. Expression of inactive variants of either PI3-K_γ, the Rac GEF Vav, or Rac blocked the actin rearrangement. Neither wortmannin nor LY294002, pharmacologic inhibitors of PI3-K, could inhibit the actin rearrangement induced by a constitutively active Rac. The inhibition of cytoskeletal reorganization by the dominant negative Vav variants could be rescued by coexpression of a constitutively active form of Rac. In contrast, a Vav variant with its pleckstrin homology (PH) domain missing constitutively induced JNK activation and led to cytoskeletal reorganization, even without stimulation by PI3-K_γ. This suggests that the PH domain of Vav controls the guanosine exchange activity of Vav, perhaps by a mechanism regulated by D3 phosphoinositides generated by PI3-K. Taken together, these findings delineate a pathway leading from activation of a G protein-coupled receptor to actin reorganization which sequentially involves G_βγ, PI3-K_γ, a Rac GEF, and Rac.     

Structural Basis of G Protein-coupled Receptor-Gi Protein Interaction: FORMATION OF THE CANNABINOID CB2 RECEPTOR-Gi PROTEIN COMPLEX*

In this study, we applied a comprehensive G protein-coupled receptor-Gα_i protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB₂)- Gα_i interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gα_i C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gα_i C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gα_i α₄β₆ loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB₂·G_i complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB₂ R*·Gα_i1β₁γ₂ complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gα_sβ₁γ₂ complex crystal structure, the Gα_i1β₁γ₂ protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gα_i1 α5 helix tilt changed due to the outward movement of TMH5 in CB₂ R*. 2) A 25° clockwise rotation of Gα_i1β₁γ₂ underneath CB₂ R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gα_i1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to G_i proteins.     

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gα_i/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gα_i1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gα_i1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gα_o-AlF₄⁻. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gα_i1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gα_o-AlF₄⁻ and an AlF₄⁻-insensitive mutant (G42R) of Gα_i1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gα_i1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gα_o-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.     

Active Detergent-solubilized H+,K+-ATPase Is a Monomer

The H⁺,K⁺-ATPase pumps protons or hydronium ions and is responsible for the acidification of the gastric fluid. It is made up of an α-catalytic and a β-glycosylated subunit. The relation between cation translocation and the organization of the protein in the membrane are not well understood. We describe here how pure and functionally active pig gastric H⁺,K⁺-ATPase with an apparent Stokes radius of 6.3 nm can be obtained after solubilization with the non-ionic detergent C₁₂E₈, followed by exchange of C₁₂E₈ with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the β-subunit bears an excess mass of 9 kDa attributable to glycosylation. From chemical analysis, there are 0.25 g of phospholipids and around 0.024 g of cholesterol bound per g of protein. Analytical ultracentrifugation shows one main complex, sedimenting at s_20,w = 7.2 ± 0.1 S, together with minor amounts of irreversibly aggregated material. From these data, a buoyant molecular mass is calculated, corresponding to an H⁺,K⁺-ATPase α,β-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an α,β-protomer with 0.9–1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of 1.5, corresponding to a Stokes radius of 7.1 nm. An α₂,β₂ dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H⁺,K⁺-ATPase. Thus, α,β H⁺,K⁺-ATPase is active at least in detergent and may plausibly function as a monomer, as has been established for other P-type ATPases, Ca²⁺-ATPase and Na⁺,K⁺-ATPase.     

A Membrane-proximal,C-terminal α-Helix Is Required for Plasma Membrane Localization and Function of the G Protein-coupled Receptor (GPCR) TGR5

The C terminus of G protein-coupled receptors (GPCRs) is important for G protein-coupling and activation; in addition, sorting motifs have been identified in the C termini of several GPCRs that facilitate correct trafficking from the endoplasmic reticulum to the plasma membrane. The C terminus of the GPCR TGR5 lacks any known sorting motif such that other factors must determine its trafficking. Here, we investigate deletion and substitution variants of the membrane-proximal C terminus of TGR5 with respect to plasma membrane localization and function using immunofluorescence staining, flow cytometry, and luciferase assays. Peptides of the membrane-proximal C-terminal variants are subjected to molecular dynamics simulations and analyzed with respect to their secondary structure. Our results reveal that TGR5 plasma membrane localization and responsiveness to extracellular ligands is fostered by a long (≥ 9 residues) α-helical stretch at the C terminus, whereas the presence of β-strands or only a short α-helical stretch leads to retention in the endoplasmic reticulum and a loss of function. As a proof-of-principle, chimeras of TGR5 containing the membrane-proximal amino acids of the β₂ adrenergic receptor (β₂AR), the sphingosine 1-phosphate receptor-1 (S1P1), or the κ-type opioid receptor (κOR) were generated. These TGR5β₂AR, TGR5S1P1, or TGR5κOR chimeras were correctly sorted to the plasma membrane. As the exchanged amino acids of the β₂AR, the S1P1, or the κOR form α-helices in crystal structures but lack significant sequence identity to the respective TGR5 sequence, we conclude that the secondary structure of the TGR5 membrane-proximal C terminus is the determining factor for plasma membrane localization and responsiveness towards extracellular ligands.     

Rab8 Interacts with Distinct Motifs in α2B- and β2-Adrenergic Receptors and Differentially Modulates Their Transport

The molecular mechanism underlying the post-Golgi transport of G protein-coupled receptors (GPCRs) remains poorly understood. Here we determine the role of Rab8 GTPase, which modulates vesicular protein transport between the trans-Golgi network (TGN) and the plasma membrane, in the cell surface targeting of α_2B- and β₂-adrenergic receptors (AR). Transient expression of GDP- and GTP-bound Rab8 mutants and short hairpin RNA-mediated knockdown of Rab8 more potently inhibited the cell surface expression of α_2B-AR than β₂-AR. The GDP-bound Rab8(T22N) mutant attenuated ERK1/2 activation by α_2B-AR, but not β₂-AR, and arrested α_2B-AR in the TGN compartment. Co-immunoprecipitation revealed that both α_2B-AR and β₂-AR physically interacted with Rab8 and glutathione S-transferase fusion protein pulldown assays demonstrated that Rab8 interacted with the C termini of both receptors. Interestingly, mutation of the highly conserved membrane-proximal C terminus dileucine motif selectively blocked β₂-AR interaction with Rab8, whereas mutation of residues Val⁴³¹-Phe⁴³²-Asn⁴³³-Gln⁴³⁴, Pro⁴⁴⁷-Trp⁴⁴⁸, Gln⁴⁵⁰-Thr⁴⁵¹, and Trp⁴⁵³ in the C terminus impaired α_2B-AR interaction with Rab8. Furthermore, transport inhibition by Rab8(T22N) of a chimeric β₂-AR carrying the α_2B-AR C terminus was similar to α_2B-AR. These data provide strong evidence indicating that Rab8 GTPase interacts with distinct motifs in the C termini of α_2B-AR and β₂-AR and differentially modulates their traffic from the TGN to the cell surface.     

Quantification of Beta Adrenergic Receptor Subtypes in Beta-Arrestin Knockout Mouse Airways

In allergic asthma Beta 2 adrenergic receptors (β₂ARs) are important mediators of bronchorelaxation and, paradoxically, asthma development. This contradiction is likely due to the activation of dual signaling pathways that are downstream of G proteins or β-arrestins. Our group has recently shown that β-arrestin-2 acts in its classical role to desensitize and constrain β₂AR-induced relaxation of both human and murine airway smooth muscle. To assess the role of β-arrestins in regulating β₂AR function in asthma, we and others have utilized β-arrestin-1 and -2 knockout mice. However, it is unknown if genetic deletion of β-arrestins in these mice influences β₂AR expression in the airways. Furthermore, there is lack of data on compensatory expression of βAR subtypes when either of the β-arrestins is genetically deleted, thus necessitating a detailed βAR subtype expression study in these β-arrestin knockout mice. Here we standardized a radioligand binding methodology to characterize and quantitate βAR subtype distribution in the airway smooth muscle of wild-type C57BL/6J and β-arrestin-1 and β-arrestin-2 knockout mice. Using complementary competition and single-point saturation binding assays we found that β₂ARs predominate over β₁ARs in the whole lung and epithelium-denuded tracheobronchial smooth muscle of C57BL/6J mice. Quantification of βAR subtypes in β-arrestin-1 and β-arrestin-2 knockout mouse lung and epithelium-denuded tracheobronchial tissue showed that, similar to the C57BL/6J mice, both knockouts display a predominance of β₂AR expression. These data provide further evidence that β₂ARs are expressed in greater abundance than β₁ARs in the tracheobronchial smooth muscle and that loss of either β-arrestin does not significantly affect the expression or relative proportions of βAR subtypes. As β-arrestins are known to modulate β₂AR function, our analysis of βAR subtype expression in β-arrestin knockout mice airways sets a reference point for future studies exploiting these knockout mice in various disease models including asthma.     

Forcing Switch from Short- to Intermediate- and Long-lived States of the αA Domain Generates LFA-1/ICAM-1 Catch Bonds

Binding of lymphocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule-1 (ICAM-1) mediates leukocyte adhesion under force. Using a biomembrane force probe capable of measuring single bond interactions, we showed ICAM-1 binding to LFA-1 at different conformations, including the bent conformation with the lowest affinity. We quantify how force and conformations of LFA-1 regulate its kinetics with ICAM-1. At zero-force, on-rates were substantially changed by conditions that differentially favor a bent or extended LFA-1 with a closed or open headpiece; but off-rates were identical. With increasing force, LFA-1/ICAM-1 bond lifetimes (reciprocal off-rates) first increased (catch bonds) and then decreased (slip bonds). Three states with distinct off-rates were identified from lifetime distributions. Force shifted the associated fractions from the short- to intermediate- and long-lived states, producing catch bonds at low forces, but increased their off-rates exponentially, converting catch to slip bonds at high forces. An internal ligand antagonist that blocks pulling of the α₇-helix suppressed the intermediate-/long-lived states and eliminated catch bonds, revealing an internal catch bond between the αA and βA domains. These results elucidate an allosteric mechanism for the mechanochemistry of LFA-1/ICAM-1 binding.     

Identifying Ligand Binding Conformations of the β2-Adrenergic Receptor by Using Its Agonists as Computational Probes

Recently available G-protein coupled receptor (GPCR) structures and biophysical studies suggest that the difference between the effects of various agonists and antagonists cannot be explained by single structures alone, but rather that the conformational ensembles of the proteins need to be considered. Here we use an elastic network model-guided molecular dynamics simulation protocol to generate an ensemble of conformers of a prototypical GPCR, β₂-adrenergic receptor (β₂AR). The resulting conformers are clustered into groups based on the conformations of the ligand binding site, and distinct conformers from each group are assessed for their binding to known agonists of β₂AR. We show that the select ligands bind preferentially to different predicted conformers of β₂AR, and identify a role of β₂AR extracellular region as an allosteric binding site for larger drugs such as salmeterol. Thus, drugs and ligands can be used as “computational probes” to systematically identify protein conformers with likely biological significance.