The Oxygen Receptor of the Teleost Gill?

The gills of the Atlantic cod, Gadus morhua, were studied using immunohistochemical techniques. Primary antibodies directed against serotonin (5-hydroxytryptamine, 5-HT) and acetylated α-tubulin were used to visualise cells containing serotonin and nerve fibres, respectively. Three morphologically different 5-HT immunoreactive cell types were distinguished: (I) Neuroepithelial cells (NECs), which were abundant along the distal half of the efferent filamental arteries (EFAs), and particularly formed distinct clusters at the individual filamental tips, (II) bipolar neurones running next to the EFAs and (III) multipolar neurones innervating the proximal parts of the EFA. In addition, the study revealed a well-developed system of nerve fibres, some of which form plexuses in association with the NECs. A relatively rich innervation of the proximal part of the EFAs, in conjunction with the EFA sphincters was also observed. Delicate varicose terminals surround the bases of the efferent lamellar arterioles. The localisation of distinct clusters of NECs at the individual filamental tips and the close connection with nerve terminals suggests a function as external branchial oxygen receptors.     

Spectroscopy-Based Modelling of the 3D Structure of the β Subunit of the High Affinity IgE Receptor

Abstract

The high affinity IgE receptor, possesses a tetrameric structure. The 243 residue β subunit is a polytopic protein with four hydrophobic membrane-spanning segments, whereas the individual α and γ subunits are bitopic proteins each containing one transmembrane domain in their monomeric form. In the proposed topographical model (Blank et al., 1989), the four trans-membrane α helices of the β subunit are connected by three loop sequences.

To study the individual subunits and intact receptor, this membrane protein was divided into domains such as its loop peptides, cytoplasmic peptides and transmembrane helices according to Blank et al., 1989. The 3D structure of the synthesized loop peptides and cytoplasmic peptides were calculated; CD and/or NMR data were used as appropriate to generate the resultant structures which were then used as data basis for the higher level calculations.

The four individual transmembrane helices of the β subunit were characterised, first of all, by mapping the relative lipophilicity of their surfaces using lipophilic probes. A second procedure, docking of the individual helices in pairs, was used to predict helix–helix interactions.

The data on the relative lipophilicity of the surfaces as well as the surfaces that favoured helix–helix interactions were used in combination with the spectroscopy-based structures of the loops and cytoplasmic domains to calculate via molecular dynamics, the helix arrangement and 3D structure of the β subunit of the high affinity IgE receptor. In the final analysis, the molecular simulations yielded two structures of the β subunit, which should form a basis for the modelling of the whole high affinity IgE receptor.     

Functional Receptor Coupling to Gi Is A Mechanism of Agonist-Promoted Desensitization of the β2-Adrenergic Receptor

Abstract

The β₂-adrenergic receptor (β₂AR) couples to G_s, activating adenylyl cyclase (AC) and increasing cAMP. Such signaling undergoes desensitization with continued agonist exposure. β₂AR also couple to G_i after receptor phosphorylation by the cAMP dependent protein kinase A, but the efficiency of such coupling is not known. Given the PKA dependence of β₂AR-G_i coupling, we explored whether this may be a mechanism of agonist-promoted desensitization. HEK293 cells were transfected to express β₂AR or β₂AR and G_iα2, and then treated with vehicle or the agonist isoproterenol to evoke agonist-promoted β₂AR desensitization. Membrane AC activities showed that G_iα2 overexpression decreased basal levels, but the fold-stimulation of the AC over basal by agonist was not altered. However, with treatment of the cells with isoproterenol prior to membrane preparation, a marked decrease in agonist-stimulated AC was observed with the cells overexpressing G_iα2. in the absence of such overexpression, β₂AR desensitization was 23 ± 7%, while with 5-fold G_iα2 overexpression desensitization was 58 ± 5% (p<0.01, n=4). the effect of G_i on desensitization was receptor-specific, in that forskolin responses were not altered by G_iα2 overexpression. Thus, acquired β₂AR coupling to G_i is an important mechanism of agonist-promoted desensitization, and pathologic conditions that increase G_i levels contribute to β₂AR dysfunction.     

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.     

Altered Localization of the δ Subunit of the GABAA Receptor in the Thalamus of α4 Subunit Knockout Mice

The α4 subunit of the GABA_A receptor (GABA_AR) is highly expressed in the thalamus where receptors containing the α4 and δ subunits are major mediators of tonic inhibition. The α4 subunit also exhibits considerable plasticity in a number of physiological and pathological conditions, raising questions about the expression of remaining GABA_AR subunits when the α4 subunit is absent. Immunohistochemical studies of an α4 subunit knockout (KO) mouse revealed a substantial decrease in δ subunit expression in the ventrobasal nucleus of the thalamus as well as other forebrain regions where the α4 subunit is normally expressed. In contrast, several subunits associated primarily with phasic inhibition, including the α1 and γ2 subunits, were moderately increased. Intracellular localization of the δ subunit was also altered. While δ subunit labeling was decreased within the neuropil, some labeling remained in the cell bodies of many neurons in the ventrobasal nucleus. Confocal microscopy demonstrated co-localization of this labeling with an endoplasmic reticulum marker, and electron microscopy demonstrated increased immunogold labeling near the endoplasmic reticulum in the α4 KO mouse. These results emphasize the strong partnership of the δ and α4 subunit in the thalamus and suggest that the α4 subunit of the GABA_AR plays a critical role in trafficking of the δ subunit to the neuronal surface. The findings also suggest that previously observed reductions in tonic inhibition in the α4 subunit KO mouse are likely to be related to alterations in δ subunit expression, in addition to loss of the α4 subunit.     

The Pharmacology of the Benzodiazepine Site of the GABA-A Receptor is Dependent on the Type of γ-Subunit Present

Abstract

The pharmacology of native and recombinant GABA-A receptors containing either γ1, γ2 or γ3 subunits has been investigated. The pharmacology of native receptors has been investigated by immunoprecipitating receptors from solubilised preparations of rat brain with antisera specific for individual γ-subunits and analysing their radioligand binding characteristics. Receptors containing a γ1-subunit do not bind benzodiazepine radioligands with high affinity. Those containing either a γ2 or γ3 subunit bind [³H]flumazenil with high affinity. Some compounds compete for these binding sites with multiple affinities, reflecting the presence of populations of receptors containing several different types of α-subunit. Photoaffinity-labelling of GABA-A receptors from a cell line stably expressing GABA-A receptors of composition α₁β3γ2 followed by immunoprecipitation of individual subunits revealed that the α and γ but not the β-subunit could be irreversibly labelled by [³H]flunitrazepam.

The properties of recombinant receptors have been investigated in oocytes expressing γ1, γ2, or γ3 subunits in combination with an α and a β-subunit. Some compounds such as zolpidem, DMCM and flunitrazepam show selectivity for receptors containing different γ-subunits. Others such as CL 218,872 show no selectivity between receptors containing different γ-subunits but exhibit selectivity for receptors containing different α-subunits. These data taken together suggest that the benzodiazepine site of the GABA-A receptor is formed with contributions from both the α and γ-subunits.     

An Alternative Conformation of the T-Cell Receptor α Constant Region

αβ T-cell receptors (TcRs) play a central role in cellular immune response. They are members of the Ig superfamily, with extracellular regions of the α and β chains each comprising a V-type domain and a C-type domain. We have determined the ectodomain structure of an αβ TcR, which recognizes the autoantigen myelin basic protein. The 2.0-Å-resolution structure reveals canonical main-chain conformations for the V^α, V^β, and C^β domains, but the C^α domain exhibits a main-chain conformation remarkably different from those previously reported for TcR crystal structures. The global IgC-like fold is maintained, but a piston-like rearrangement between BC and DE β-turns results in β-strand slippage. This substantial conformational change may represent a signaling intermediate. Our structure is the first example for the Ig fold of the increasingly recognized concept of “metamorphic proteins.”     

Functional Characteristics of the Naked Mole Rat μ-Opioid Receptor

While humans and most animals respond to µ-opioid receptor (MOR) agonists with analgesia and decreased aggression, in the naked mole rat (NMR) opioids induce hyperalgesia and severe aggression. Single nucleotide polymorphisms in the human mu-opioid receptor gene (OPRM1) can underlie altered behavioral responses to opioids. Therefore, we hypothesized that the primary structure of the NMR MOR may differ from other species. Sequencing of the NMR oprm1 revealed strong homology to other mammals, but exposed three unique amino acids that might affect receptor-ligand interactions. The NMR and rat oprm1 sequences were cloned into mammalian expression vectors and transfected into HEK293 cells. Radioligand binding and 3''-5''-cyclic adenosine monophosphate (cAMP) enzyme immunoassays were used to compare opioid binding and opioid-mediated cAMP inhibition. At normalized opioid receptor protein levels we detected significantly lower [3H]DAMGO binding to NMR compared to rat MOR, but no significant difference in DAMGO-induced cAMP inhibition. Strong DAMGO-induced MOR internalization was detectable using radioligand binding and confocal imaging in HEK293 cells expressing rat or NMR receptor, while morphine showed weak or no effects. In summary, we found minor functional differences between rat and NMR MOR suggesting that other differences e.g. in anatomical distribution of MOR underlie the NMR''s extreme reaction to opioids.     

A Residue in Loop 9 of the ��2-Subunit Stabilizes the Closed State of the GABAA Receptor

In γ-aminobutyric acid type A (GABA_A) receptors, the structural elements that couple ligand binding to channel opening remain poorly defined. Here, site-directed mutagenesis was used to determine if Loop 9 on the non-GABA binding site interface of the β2-subunit may be involved in GABA_A receptor activation. Specifically, residues Gly¹⁷⁰-Gln¹⁸⁵ of the β2-subunit were mutated to alanine, co-expressed with wild-type α1- and γ2S-subunits in human embryonic kidney (HEK) 293 cells and assayed for their activation by GABA, the intravenous anesthetic propofol and the endogenous neurosteroid pregnanolone using whole cell macroscopic recordings. Three mutants, G170A, V175A, and G177A, produced 2.5-, 6.7-, and 5.6-fold increases in GABA EC₅₀ whereas one mutant, Q185A, produced a 5.2-fold decrease in GABA EC₅₀. None of the mutations affected the ability of propofol or pregnanolone to potentiate a submaximal GABA response, but the Q185A mutant exhibited 8.3- and 3.5-fold increases in the percent direct activation by propofol and pregnanolone, respectively. Mutant Q185A receptors also had an increased leak current that was sensitive to picrotoxin, indicating an increased gating efficiency. Further Q185E, Q185L, and Q185W substitutions revealed a strong correlation between the hydropathy of the amino acid at this position and the GABA EC₅₀. Taken together, these results indicate that β2 Loop 9 is involved in receptor activation by GABA, propofol, and pregnanolone and that β2(Q185) participates in hydrophilic interactions that are important for stabilizing the closed state of the GABA_A receptor.     

Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC₅₀ to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of ¹²⁵I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

Receptor cells in the pineal organ of the dogfish Scyliorhinus canicula linné

Summary The pineal parenchyma of the dogfish Scyliorhinus canicula contains sensory (receptor) cells and supporting cells. The ultrastructural characteristics of these cells are described. The sensory cell is a photoreceptor-like cell the outer segment of which is, however, often irregularly developed. Neuropil-like areas are present but no typical synapses have been observed. The classification of pineal receptor cells is discussed.Work done with the aid of a research scholarship from the Alexander von Humboldt Foundation, Bad Godesberg, Germany. — The electron microscope used in this study was placed at the disposal of Professor Oksche by the Deutsche Forschungsgemeinschaft. — The animal material was provided by the Stazione Zoologica di Napoli, Italy.     

Roles of the Adenosine Receptor and CD73 in the Regulatory Effect of γδ T Cells

The adenosine A2A receptor (A2AR), the main functional adenosine receptor on murine T cells, plays a unique role in the attenuation of inflammation and tissue damage in vivo. Here, we showed that, of the immune cell types tested, activated γδ T cells expressed the highest levels of A2AR mRNA and that A2AR ligation inhibited αβ T cell activation, but enhanced γδ T cell activation. We also showed that the inhibitory effect of an adenosine receptor agonist on autoreactive T cells was prevented by addition of a low percentage of activated γδ T cells. Furthermore, compared to resting cells, activated γδ T cells expressed significantly lower levels of CD73, an enzyme involved in the generation of extracellular adenosine. Exogenous AMP had a significant inhibitory effect on autoreactive T cell responses, but only in the presence of CD73⁺ γδ T cells, and this effect was abolished by a CD73 inhibitor. Our results show that expression of increased amounts of A2AR allows γδ T cells to bind adenosine and thereby attenuate its suppressive effect, while decreased expression of CD73 results in less generation of adenosine in the inflammatory site. Together, these events allow activated γδ T cells to acquire increased proinflammatory activity, leading to augmented autoimmune responses.     

Inactivation of the Purified Bovine μ Opioid Receptor by Sulfhydryl Reagents

We have investigated the role of cysteine residues in a highly purified opioid receptor protein (ORP) by examining the effect of -SH reagents on the binding of opioid ligands. Treatment of ORP, which is devoid of additional proteins, eliminates complications that arise from reaction of -SH reagents with other components, such as G proteins. Reagents tested include N-ethylmaleimide, 5,5-dithiobis(2-nitrobenzoic) acid, and two derivatives of methanethiosulfonate. Specific opioid binding was inactivated by micromolar concentrations of all -SH reagents tested. Agonist binding ([³H]DAMGO) was much more sensitive to inactivation than antagonist binding ([³H]bremazocine). Prebinding ORP with 100 nM naloxone protected antagonist and agonist binding from inactivation by -SH reagents. The results of these experiments strongly suggest that at least one, and possibly more, reactive cysteine residue(s) is present on the opioid receptor protein molecule, positioned near the ligand binding site and accessible to -SH reagents.     

Prostaglandin E2 Stimulates the Production of Amyloid-�� Peptides through Internalization of the EP4 Receptor

Amyloid-β (Aβ) peptides, generated by the proteolysis of β-amyloid precursor protein by β- and γ-secretases, play an important role in the pathogenesis of Alzheimer disease. Inflammation is also important. We recently reported that prostaglandin E₂ (PGE₂), a strong inducer of inflammation, stimulates the production of Aβ through EP₂ and EP₄ receptors, and here we have examined the molecular mechanism. Activation of EP₂ and EP₄ receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP₂, but not EP₄, receptor-mediated stimulation of the Aβ production. In contrast, inhibitors of endocytosis suppressed EP₄, but not EP₂, receptor-mediated stimulation. Activation of γ-secretase was observed with the activation of EP₄ receptors but not EP₂ receptors. PGE₂-dependent internalization of the EP₄ receptor was observed, and cells expressing a mutant EP₄ receptor lacking the internalization activity did not exhibit PGE₂-stimulated production of Aβ. A physical interaction between the EP₄ receptor and PS-1, a catalytic subunit of γ-secretases, was revealed by immunoprecipitation assays. PGE₂-induced internalization of PS-1 and co-localization of EP₄, PS-1, and Rab7 (a marker of late endosomes and lysosomes) was observed. Co-localization of PS-1 and Rab7 was also observed in the brain of wild-type mice but not of EP₄ receptor null mice. These results suggest that PGE₂-stimulated production of Aβ involves EP₄ receptor-mediated endocytosis of PS-1 followed by activation of the γ-secretase, as well as EP₂ receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of Alzheimer disease.Alzheimer disease (AD)² is the most common neurodegenerative disorder of the central nervous system and the leading cause of adult onset dementia. AD is characterized pathologically by the accumulation of tangles and senile plaques. Senile plaques are composed of the amyloid-β (Aβ) peptides Aβ40 and Aβ42 (1, 2). To generate Aβ, β-amyloid precursor protein (APP) is first cleaved by β-secretase and then by γ-secretase. Cleavage of APP by α-secretase produces non-amyloidogenic peptides (3, 4). The γ-secretase is an aspartyl protease complex composed of four core components, including presenilin (PS) 1 and PS2 (5). Early onset familial AD is linked to three genes, APP, PS1, and PS2 (5, 6), strongly suggesting that γ-secretase-dependent production of Aβ is a key factor in the pathogenesis of AD. Therefore, cellular factors that affect the γ-secretase-dependent production of Aβ may be good targets for the development of drugs to prevent and treat AD.Both APP and PS-1 are transmembrane proteins, and their intracellular localization is controlled by secretory and endocytic pathways. These proteins are modified in the endoplasmic reticulum and trafficked to the cell surface through the trans-Golgi network (TGN). Then, they are internalized again and trafficked to early endosomes. Next, they are trafficked to late endosomes and lysosomes (LEL), which are recycling endosomes that are targeted to the cell surface or the TGN (7–11). The production of Aβ seems to occur in a broad range of cellular compartments including the cell surface, TGN, and endosomes (12). Abnormalities of secretory and endocytic pathways have been observed in the brains of AD patients (9, 13). Importantly, factors that control these vesicle transport systems affect the production of Aβ. For example, overproduction of Rab5, a factor essential for traffic of vesicles to early endosomes, has been shown to stimulate the production of Aβ (14), and SorL1 has been shown to reduce the production of Aβ by stimulating the traffic of APP in early endosomes to the TGN (15, 16).It has been suggested that inflammation is important in the pathogenesis of AD; chronic inflammation has been observed in the brains of AD patients, and trauma to the brain and ischemia, both of which can activate inflammation, are major risk factors for AD (17–19). Cyclooxygenase (COX) is essential for the synthesis of prostaglandin E₂ (PGE₂), a potent inducer of inflammation and has two subtypes, COX-1 and COX-2. COX-1 is expressed constitutively, whereas expression of COX-2 is induced under inflammatory conditions and is responsible for the progression of inflammation (20–22). The following evidences of the involvement of PGE₂ (and COX-2) in the progression of AD suggest that they are good targets for the development of AD drugs: (i) Elevated levels of PGE₂ and overexpression of COX-2 have been observed in the brains of AD patients (23–25); (ii) the extent of COX-2 expression correlates with the amount of Aβ and the degree of progression of AD pathogenesis (26); (iii) transgenic mice constitutively overexpressing COX-2 show aging-dependent neural apoptosis and memory dysfunction (27); (iv) prolonged use of nonsteroidal anti-inflammatory drugs, inhibitors of COX, delays the onset and reduces the risk of AD (28); (v) PGE₂ stimulates the production of reactive oxygen species in microglia cells, resulting in activation of β-secretase (29).We recently reported that PGE₂ stimulates the production of Aβ in human embryonic kidney (HEK) 293 and human neuroblastoma (SH-SY5Y) cells that stably express a form of APP with two mutations (K651N/M652L) (APPsw) that elevate cellular and secreted levels of Aβ (30). Similar results were reported by another group (31). Using agonists and antagonists specific for each of the four PGE₂ receptors (EP₁, EP₂, EP₃, and EP₄), we found that EP₄ receptors alone and also both EP₂ and EP₄ receptors are involved in PGE₂-stimulated production of Aβ in HEK293 or SH-SY5Y cells, respectively (30). Furthermore, experiments with transgenic mice suggest that EP₂ and EP₄ receptors are involved in the production of Aβ in vivo (30). Based on these results, we propose that antagonists of the EP₂ and/or EP₄ receptors may be therapeutically beneficial for the treatment of AD. Understanding the mechanism governing EP₂ and EP₄ receptor-mediated stimulation of production of Aβ by PGE₂ will be important for such drug development.Activation of EP₂ and EP₄ receptors causes activation of adenylate cyclase and an increase in the cellular level of cAMP (32). We have shown that an EP₄ receptor agonist or both EP₂ and EP₄ receptor agonists increase the cellular level of cAMP in HEK293 or SH-SY5Y cells, respectively, and that a cAMP analogue, 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP), increases the level of Aβ in HEK293 cells (30). These findings suggest that the cellular level of cAMP is important for PGE₂-stimulated production of Aβ. An increase in the cellular level of cAMP is known to activate protein kinase A (PKA), which is important for cAMP-regulated intracellular signal transduction (33). However, an inhibitor of PKA, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89), does not block PGE₂-stimulated production of Aβ in HEK293 cells (30). Other cAMP-regulated signal transduction factors, such as phosphatidylinositol 3-kinase and Epac (exchange protein directly activated by cAMP), were also shown not to be involved in PGE₂-stimulated production of Aβ in HEK293 cells (30). Thus, the mechanism whereby the activation of EP₂ and EP₄ receptors stimulates the production of Aβ has remained unknown. In this study, by using inhibitors of adenylate cyclase and PKA, we found that activation of the EP₂ receptor stimulates production of Aβ through activation of adenylate cyclase and PKA. We also propose that activation of the EP₄ receptor causes its co-internalization with PS-1 (γ-secretase) into endosomes and that this co-internalization is important for EP₄ receptor-mediated stimulation of Aβ production by PGE₂ through the activation of γ-secretase.     

Molecular Insights into the Dynamics of Pharmacogenetically Important N-Terminal Variants of the Human β2-Adrenergic Receptor

The human β₂-adrenergic receptor (β₂AR), a member of the G-protein coupled receptor (GPCR) family, is expressed in bronchial smooth muscle cells. Upon activation by agonists, β₂AR causes bronchodilation and relief in asthma patients. The N-terminal polymorphism of β₂AR at the 16^th position, Arg16Gly, has warranted a lot of attention since it is linked to variations in response to albuterol (agonist) treatment. Although the β₂AR is one of the well-studied GPCRs, the N-terminus which harbors this mutation, is absent in all available experimental structures. The goal of this work was to study the molecular level differences between the N-terminal variants using structural modeling and atomistic molecular dynamics simulations. Our simulations reveal that the N-terminal region of the Arg variant shows greater dynamics than the Gly variant, leading to differential placement. Further, the position and dynamics of the N-terminal region, further, affects the ligand binding-site accessibility. Interestingly, long-range effects are also seen at the ligand binding site, which is marginally larger in the Gly as compared to the Arg variant resulting in the preferential docking of albuterol to the Gly variant. This study thus reveals key differences between the variants providing a molecular framework towards understanding the variable drug response in asthma patients.     

Activation of the B Cell Receptor Leads to Increased Membrane Proximity of the Igα Cytoplasmic Domain

Binding of antigen to the B cell receptor (BCR) induces conformational changes in BCR''s cytoplasmic domains that are concomitant with phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs). Recently, reversible folding of the CD3ε and ξ chain ITAMs into the plasma membrane has been suggested to regulate T cell receptor signaling. Here we show that the Igα and Igβ cytoplasmic domains of the BCR do not associate with plasma membrane in resting B cells. However, antigen binding and ITAM phosphorylation specifically increased membrane proximity of Igα, but not Igβ. Thus, BCR activation is accompanied by asymmetric conformational changes, possibly promoting the binding of Igα and Igβ to differently localized signaling complexes.