Multiple activation steps of the N-formyl peptide receptor

The human N-formyl peptide receptor (FPR) is representative of a growing family of G protein-coupled receptors (GPCR) that respond to chemokines and chemoattractants. Despite the importance of this receptor class to immune function, relatively little is known about the molecular mechanisms involved in their activation. To reveal steps required for the activation of GPCR receptors, we utilized mutants of the FPR which have previously been shown to be incapable of binding and activating G proteins. For this study, the FPR mutants were expressed in human myeloid U937 cells and characterized for functions in addition to G protein coupling, such as receptor phosphorylation and ligand-induced receptor internalization. The results demonstrated that one of the mutants, R123G, though being unable to activate G protein, was capable of undergoing ligand-induced phosphorylation as well as internalization. Receptor internalization was monitored by following the fate of the ligand as well as by directly monitoring the fate of the receptor. The results with the R123G mutant were in contrast to those obtained for mutants D71A and R309G/E310A/R311G which, though being expressed at the cell surface and binding ligand, were incapable of being phosphorylated or internalized upon agonist stimulation. These results suggest that following ligand binding at least two "steps" are required for full activation of the wild-type FPR. That these observations may be of more general importance in GPCR-mediated signaling is suggested by the highly conserved nature of the mutants studied: D71, R123, and the site represented by amino acids 309-311 are very highly conserved throughout the entire superfamily of G protein-coupled receptors. Models of receptor activation based on the observed results are discussed.     

Physicochemical properties of the N-formyl peptide receptor on human neutrophils

In order to investigate the physicochemical properties of the N-formyl peptide receptor of human neutrophils, the receptor was specifically and covalently labeled with an iodinated, photoactivatable derivative of the chemotactic hexapeptide, N-formyl-norleucylleucyl- phenylalanyl-norleucyl-[125I]iodotyrosyl-N epsilon-(6- (4'-azido-2'-nitrophenylamino) hexanoyl)-lysine. After labeling isolated neutrophil membranes, the receptor was extracted with Triton X-100, digitonin, or octyl glucoside and subjected to gel filtration on a calibrated Ultrogel AcA 34 column. The Triton X-100- and digitonin-extracted receptor eluted as single molecular species, with Stokes radii of 40 and 33 A, respectively. This material was subjected to further physicochemical analysis. When octyl glucoside-extracted material was gel-filtered, a second peak containing specifically labeled material eluted in the void volume. Subsequent sodium dodecyl sulfate-polyacryl-amide amide gel electrophoresis analysis indicated that this species was the result of disulfide bonded aggregates containing the monomeric species. Sedimentation equilibrium analysis was carried out in H2O and D2O/H2O mixtures, yielding an apparent molecular mass of 63,000 daltons for both Triton X-100- and digitonin-extracted receptor. This agrees closely with the reduced sodium dodecyl sulfate-polyacrylamide gel electrophoretic value of 50,000-60,000 daltons, indicating that the receptor extracted from unstimulated membranes is monomeric in these detergents. From the sedimentation equilibrium data, the partial specific volume (v) and frictional ratio (f/f0) were calculated. The v is high in both Triton X-100 (0.880) and digitonin (0.829), indicating that the receptor may be associated with tightly bound endogenous lipid or that it is a hydrophobic membrane protein. This latter likelihood is further supported by the quantitative extraction of receptor into Triton X-114 by a phase-separation method. The frictional ratio of 1.1-1.3 is consistent with an elongated globular protein having an axial ratio of approximately 3:1. This in conjunction with the Stokes radius of 40 A would indicate that the receptor is capable of spanning the 35-40-A nonpolar center of the lipid bilayer. The state of the receptor in situ is discussed.     

Kinetics of N-formyl peptide receptor up-regulation during stimulation in human neutrophils

The kinetics of receptor up-regulation was examined in isolated neutrophils and in whole blood by flow cytometry during cell activation. Stimulation of neutrophils prepared without exposure to LPS with chemoattractants induced fast up-regulation of N-formyl peptide receptors and C receptor type 3 (CR3). Biphasic N-formyl peptide binding curves were detected for saturating concentrations of N-formyl peptide at 37 degrees C. The bulk of the rapid binding during the first 30 to 60 s is attributed to already expressed binding sites whereas the slow binding over the next 3 to 4 min represents a time course of receptor up-regulation. Support for this interpretation comes from conditions under which the number of binding sites and the progress of the binding curves were affected. Cells treated with LPS, which caused expression of internal N-formyl peptide receptors, exhibited rapid, monophasic binding curves with increased total binding. In LPS-untreated, calcium-depleted cells, N-formyl peptide receptor up-regulation was inhibited and rapid, monophasic binding to a smaller total number of expressed sites was observed. Cytochalasin B enhanced the total number of available N-formyl peptide receptors in LPS-untreated but not LPS-treated cells. In both cases binding was rapid and monophasic suggesting that receptors were either fully or rapidly up-regulated. Although not studied in real-time, C receptor type 3 up-regulation was similar to N-formyl peptide receptor up-regulation in response to LPS, or stimulation by N-formyl peptide, C product C5a, leukotriene B4, and platelet-activating factor in isolated cells and in whole blood. After stimulation with formyl peptide, LPS, or C product 5a, the release of vitamin B12-binding protein paralleled up-regulation of receptors. These data indicate that untreated cells up-regulate N-formyl peptide receptors during cell response at a rate of approximately 10,000/min in a calcium-dependent manner whereas LPS-treated cells already express the bulk of their receptors. In cytochalasin B-treated, degranulating cells 30,000 to 50,000 receptors were up-regulated within a minute.     

Receptor binding kinetics and cellular responses of six N-formyl peptide agonists in human neutrophils

The goal of this study was to elucidate the relationships between early ligand binding/receptor processing events and cellular responses for the N-formyl peptide receptor system on human neutrophils as a model of a GPCR system in a physiologically relevant context. Binding kinetics of N-formyl-methionyl-leucyl-phenylalanyl-phenylalanyl-lysine-fluorescein and N-formyl-valyl-leucyl-phenylalanyl-lysine-fluorescein to the N-formyl peptide receptor on human neutrophils were characterized and combined with previously published binding data for four other ligands. Binding was best fit by an interconverting two-receptor state model that included a low affinity receptor state that converted to a high affinity state. Response behaviors elicited at 37 degrees C by the six different agonists for the N-formyl peptide receptor were measured. Dose response curves for oxidant production, actin polymerization, and G-protein activation were obtained for each ligand; whereas all ligands showed equal efficacy for all three responses, the ED(50) values varied as much as 7000-fold. The level of agonism and rank order of potencies of ligands for actin and oxidant responses were the same as for the G-protein activation assay, suggesting that the differences in abilities of ligands to mediate responses were determined upstream of G-protein activation at the level of ligand-receptor interactions. The rate constants governing ligand binding and receptor affinity conversion were ligand-dependent. Analysis of the forward and reverse rate constants governing binding to the proposed signaling receptor state showed that it was of a similar energy for all six ligands, suggesting the hypothesis that ligand efficacy is dictated by the energy state of this ligand-receptor complex. However, the interconverting two-receptor state model was not sufficient to predict response potency, suggesting the presence of receptor states not discriminated by the binding data.     

Synthesis and use of a novel N-formyl peptide derivative to isolate a human N-formyl peptide receptor cDNA

N-formyl-methionyl peptides are powerful chemoattractants which bind to specific receptors on the neutrophil plasma membrane. A cDNA library from HL-60 cells, differentiated into granulocytes highly responsive to N-formyl-methionyl peptides, was constructed in the COS cell expression vector CDM8. A cDNA clone was isolated that conferred to COS cells the ability to bind a new and highly efficient hydrophilic derivative of N-formyl-Met-Leu-Phe-Lys. The transfected COS cells displayed two classes of binding sites with Kd values of 0.5-1 nM and 5-10 nM, respectively. The cDNA was 1.9 kb long with a 1050 bp open reading frame encoding a 350 residue protein. The hydropathy plot analysis revealed seven hydrophobic segments, a pattern quite similar to that of G protein-coupled receptors.     

Influence of botulinum C2 toxin on F-actin and N-formyl peptide receptor dynamics in human neutrophils

Stimulation of human neutrophils with the chemotactic N-formyl peptide causes production of oxygen radicals and conversion of monomeric actin (G-actin) to polymeric actin (F-actin). The effects of the binary botulinum C2 toxin on the amount of F-actin and on neutrophil cell responses were studied. Two different methods for analyzing the actin response were used in formyl peptide-stimulated cells: staining of F-actin with rhodamine-phalloidin and a transient right angle light scatter. Preincubation of neutrophils with 400 ng/ml component I and 1,600 ng/ml component II of botulinum C2 toxin for 30 min almost completely inhibited the formyl peptide-stimulated polymerization of G-actin and at the same time decreased the amount of F-actin in unstimulated neutrophils by an average of approximately 30%. Botulinum C2 toxin preincubation for 60 min destroyed approximately 75% of the F-actin in unstimulated neutrophils. Right angle light scatter analysis showed that control neutrophils exhibited the transient response characteristic of actin polymerization; however, after botulinum C2 toxin treatment, degranulation was detected. Single components of the binary botulinum C2 toxin were without effect on the actin polymerization response. Fluorescence flow cytometry and fluorospectrometric binding studies showed little alteration in N-formyl peptide binding or dissociation dynamics in the toxin-treated cells. However, endocytosis of the fluorescent N-formyl peptide ligand-receptor complex was slower but still possible in degranulating neutrophils treated with botulinum C2 toxin for 60 min. The half-time of endocytosis, estimated from initial rates, was 4 and 8 min in control and botulinum C2 toxin-treated neutrophils, respectively.     

gamma-Aminobutyric acid/benzodiazepine receptor protein carries binding sites for both ligands on both two major peptide subunits

Affinity column-purified GABA-benzodiazepine receptor proteins from human, cow, and rat brain were photoaffinity labeled with both [3H]flunitrazepam and [3H]muscimol and examined by gel electrophoresis in sodium dodecyl sulfate. Using high receptor protein concentrations (1 microM), the benzodiazepine ligand [3H]flunitrazepam was incorporated covalently primarily into the expected 52 kiloDalton major subunit but also significantly into a second 57 kiloDalton peptide. Likewise the GABA ligand [3H]muscimol photolabeled primarily the 57 kiloDalton peptide but also to some extent the 52 kiloDalton peptide. This cross-labeling suggests strongly that both major subunits carry binding sites for both GABA and benzodiazepine.     

Selection of peptide ligands binding to fibroblast growth factor receptor 1

Inappropriate expression of fibroblast growth factors (FGFs) or activation of FGF receptors (FGFRs) could contribute to several human angiogenic pathologies. In an attempt to design antagonists of FGF, we developed a screening procedure for identifying peptide ligands binding to FGFR1. To retain the natural conformation of FGFR1 during screening, we expressed recombinant FGFR1 on the surface of Sf9 insect cells. A 6-mer phage display peptide library was then screened on the cell surface and a group of hydrophobic peptide sequences were identified. Further experiments demonstrated that the phages displaying these sequences can specifically bind to FGFR1. The docking analysis suggests that the peptide ValTyrMetSerProPhe can specifically bind to the hydrophobic surface of FGFR1. The synthetic peptide Ac-ValTyrMetSerProPhe-NH2 can inhibit mitogenic activity of aFGF and has the potential to become a therapeutic agent as an aFGF antagonist.     

Human neutrophils are activated by a peptide fragment of Clostridium difficile toxin B presumably via formyl peptide receptor

Clostridium difficile may induce antibiotic‐associated diarrhoea and, in severe cases, pseudomembranous colitis characterized by tremendous neutrophil infiltration. All symptoms are caused by two exotoxins: TcdA and TcdB. We describe here the activation of isolated human blood neutrophils by TcdB and, moreover, by toxin fragments generated by limited proteolytical digestion. Kinetics and profiles of TcdB‐induced rise in intracellular‐free Ca²⁺ and reactive oxygen species production were similar to that induced by fMLF, which activates the formyl peptide receptor (FPR) recognizing formylated bacterial peptide sequences. Transfection assays with the FPR‐1 isoform hFPR26 in HEK293 cells, heterologous desensitization experiments and FPR inhibition via cyclosporine H strongly suggest activation of cells via FPR‐1. Domain analyses revealed that the N‐terminal glucosyltransferase domain of TcdB is a potent activator of FPR pointing towards an additional mechanism that might contribute to pathogenesis. This pro‐inflammatory ligand effect can be triggered even by cleaved and, thus, non‐cytotoxic toxin. In summary, we report (i) a ligand effect on neutrophils as completely new molecular mode of action, (ii) pathogenic potential of truncated or proteolytically cleaved ‘non‐cytotoxic’ fragments and (iii) an interaction of the N‐terminal glucosyltransferase domain instead of the C‐terminal receptor binding domain of TcdB with target cells.     

Beta-arrestin binding to the beta2-adrenergic receptor requires both receptor phosphorylation and receptor activation

Homologous desensitization of beta2-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of beta-arrestins to the phosphorylated receptor. Binding of beta-arrestin to the receptor is a prerequisite for subsequent receptor desensitization, internalization via clathrin-coated pits, and the initiation of alternative signaling pathways. In this study we have investigated the interactions between receptors and beta-arrestin2 in living cells using fluorescence resonance energy transfer. We show that (a) the initial kinetics of beta-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind beta-arrestin2 very rapidly; and (c) the interaction of beta-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-beta-arrestin2 complex. This fast agonist-controlled association and dissociation of beta-arrestins from prephosphorylated receptors should permit rapid control of receptor sensitivity in repeatedly stimulated cells such as neurons.     

Partial phosphorylation of the N-formyl peptide receptor inhibits G protein association independent of arrestin binding

It is now well accepted that G protein-coupled receptors activated by agonist binding become targets for phosphorylation, leading to desensitization of the receptor. Using a series of phosphorylation deficient mutants of the N-formyl peptide receptor (FPR), we have explored the role of phosphorylation on the ability of the receptor to interact with G proteins and arrestins. Using a fluorometric assay in conjunction with solubilized receptors, we demonstrate that phosphorylation of the wild type FPR lowers its affinity for G protein, whereas mutant receptors lacking four potential phosphorylation sites retain their ability to couple to G protein. Phosphorylated mutant receptors lacking only two potential phosphorylation sites are again unable to couple to G protein. Furthermore, whereas stimulated wild type FPR in whole cells colocalizes with arrestin-2, and the solubilized, phosphorylated FPR binds arrestin-2, the stimulated receptors lacking four potential phosphorylation sites display no interaction with arrestin-2. However, the mutant receptors lacking only two potential phosphorylation sites are restored in their ability to bind and colocalize with arrestin-2. Thus, there is a submaximal threshold of FPR phosphorylation that simultaneously results in an inhibition of G protein binding and an induction of arrestin binding. These results are the first to demonstrate that less than maximal levels of receptor phosphorylation can block G protein binding, independent of arrestin binding. We therefore propose that phosphorylation alone may be sufficient to desensitize the FPR in vivo, raising the possibility that for certain G protein-coupled receptors, desensitization may not be the primary function of arrestin.     

Shear-induced resistance to neutrophil activation via the formyl peptide receptor

The application of fluid shear stress on leukocytes is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. The formyl peptide receptor (FPR) on neutrophils, which binds to formyl-methionyl-leucyl-phenylalanine (fMLP) and plays a role in neutrophil chemotaxis, has been implicated as a fluid shear stress sensor that controls pseudopod formation. The role of shear forces on earlier indicators of neutrophil activation, such as L-selectin shedding and α(M)β(2) integrin activation, remains unclear. Here, human neutrophils exposed to uniform shear stress (0.1-4.0 dyn/cm(2)) in a cone-and-plate viscometer for 1-120 min showed a significant reduction in both α(M)β(2) integrin activation and L-selectin shedding after stimulation with 0.5 nM of fMLP. Neutrophil resistance to activation was directly linked to fluid shear stress, as the response increased in a shear stress force- and time-dependent manner. Significant shear-induced loss of FPR surface expression on neutrophils was observed, and high-resolution confocal microscopy revealed FPR internalized within neutrophils. These results suggest that physiological shear forces alter neutrophil activation via FPR by reducing L-selectin shedding and α(M)β(2) integrin activation in the presence of soluble ligand.     

Specific residues in plasmatocyte-spreading peptide are required for receptor binding and functional antagonism of insect immune cells

Plasmatocyte-spreading peptide (PSP) is a 23-amino acid cytokine that activates a class of insect immune cells called plasmatocytes. PSP consists of two regions: an unstructured N terminus (1-6) and a highly structured core (7-23). Prior studies identified specific residues in both the structured and unstructured regions required for biological activity. Most important for function were Arg13, Phe3, Cys7, Cys19, and the N-terminal amine of Glu1. Here we have built on these results by conducting cell binding and functional antagonism studies. Alanine replacement of Met12 (M12A) resulted in a peptide with biological activity indistinguishable from PSP. Competitive binding experiments using unlabeled and 125I-M12A generated an IC50 of 0.71 nm and indicated that unlabeled M12A, at concentrations > or =100 nm, completely blocked binding of label to hemocytes. We then tested the ability of other peptide mutants to displace 125I-M12A at a concentration of 100 nm. In the structured core, we found that Cys7 and Cys19 were essential for cell binding and functional antagonism, but these effects were likely because of the importance of these residues for maintaining the tertiary structure of PSP. Arg13, in contrast, was also essential for binding and activity but is not required for maintenance of structure. In the unstructured N-terminal region, deletion of the phenyl group from Phe3 yielded a peptide that reduced binding of 125I-M12A 326-fold. This and all other mutants of Phe3 we bioassayed were unable to antagonize PSP. Deletion of Glu1 in contrast had almost no effect on binding and was a strong functional antagonist. Experiments using a photoaffinity analog indicated that PSP binds to a single 190-kDa protein.     

Arrestin binding to the G protein-coupled N-formyl peptide receptor is regulated by the conserved "DRY" sequence

Following activation by ligand, the N-formyl peptide receptor (FPR) undergoes processing events initiated by phosphorylation that lead to receptor desensitization and internalization. Our previous results have shown that FPR internalization can occur in the absence of receptor desensitization, suggesting that FPR desensitization and internalization are controlled by distinct mechanisms. More recently, we have provided evidence that internalization of the FPR occurs via a mechanism that is independent of the actions of arrestin, dynamin, and clathrin. In the present report, we demonstrate that stimulation of the FPR with agonist leads to a significant translocation of arrestin-2 from the cytosol to the membrane. Fluorescence microscopy revealed that the translocated arrestin-2 is highly colocalized with the ligand-bound FPR. A D71A mutant FPR, which does not undergo activation or phosphorylation in response to ligand, did not colocalize with arrestin-2. Surprisingly, an R123G mutant FPR, which does not bind G protein but does become phosphorylated and subsequently internalized, also did not bind arrestin. These results indicate that arrestin binding is not required for FPR internalization and demonstrate for the first time that a common motif, the conserved "DRY" domain of G protein-coupled receptors, is essential for phosphorylation-dependent arrestin binding, as well as G protein activation.     

Reconstitution and characterization of the human neutrophil N-formyl peptide receptor and GTP binding proteins in phospholipid vesicles

We have developed a unilamellar phospholipid vesicle system which contains the N-formyl peptide receptor and GTP binding proteins. Several detergents were investigated but only two, octyl glucoside (35 mM) and deoxycholate (7.5 mM), were capable of extracting N-formyl peptide receptor from neutrophil membranes in a form which remained functionally active upon reconstitution into phospholipid vesicles. Extracted proteins were reconstituted into phosphatidylcholine vesicles by passage over a Sephadex G-50-80 column. The reconstituted formylpeptide receptor could bind [3H]FMLP (3H-labeled fMet-Leu-Phe) and [125I]FMLPL-SASD (125I-labeled N-formylmethionylleucylphenylalanyl-N epsilon-(2-(p-azidosalicylamido)ethyl- 1,3'-dithiopropionyl)lysine) while the endogenous G protein could bind [35S]GTP gamma S. Furthermore, the functional interaction of the two proteins was preserved. Addition of the nonhydrolyzable guanine nucleotide, GTP gamma S, shifted the N-formyl peptide receptor from a high- to a low-affinity binding state for ligand. The development of this in vitro reconstitution system should provide a basis to study the mechanism of interaction of the N-formyl peptide receptor and the G protein.     

Mu-opioid receptor and CREB activation are required for nicotine reward

Environmental cues associated with nicotine delivery are an important part of the stimulus that sustains smoking behavior and is often coupled with craving and relapse; however, the neuronal circuitry and molecular substrates underlying this process are still poorly understood. Exposure to an environment previously associated with rewarding properties of nicotine results in an increase of CREB phosphorylation similar to that seen following nicotine administration, and this response is absent in MOR(-/-) mice. Moreover, a single administration of an opioid receptor antagonist, naloxone, blocks both the conditioned molecular response (CREB phosphorylation) and the conditioned behavioral response (nicotine reward) in a place preference paradigm. Lastly, repeated nicotine administration results in increased expression of MORs. However, this effect, along with rewarding properties of nicotine, is blocked in mice with a targeted disruption in the CREB gene. Together, pharmacologic and genetic manipulations indicate that phosphorylation of CREB and upregulation of functional MORs are required for nicotine-conditioned reward.     

Ligand/receptor internalization: a kinetic, flow cytometric analysis of the internalization of N-formyl peptides by human neutrophils

Fluorescence flow cytometry was used to measure the internalization of the fluorescent ligand N-formyl-nle-leu-phe-nle-tyr-lys-fluorescein by human neutrophils. The internalization process was monitored by the accessibility of the receptor-bound fluorescent ligand to quenching following a change in the pH of the extracellular medium from 7.4 to 3.0. In such a pH change, extracellular ligand or fluorescein are quenched immediately (excitation 488 nm). In contrast, intracellular fluorescein (derived from fluorescein diacetate) or intracellular ligand are quenched with half-times of approximately 20 or approximately 40 sec, respectively, at 37 degrees C. The fraction of internalized ligand is calculated by resolving the fast and slow components of the quenching process. Temporal resolution of the internalization process in this system depends upon two factors. We have previously shown that it is possible to examine essentially continuously the kinetics of ligand binding in the nM concentration range without removing the free ligand (Sklar LA, Finney DA, Cytometry 3:161, 1982). We have now modified a Becton Dickinson FACS IV sample head assembly to permit direct addition of reagents into the cell suspension while on-line. This enables us to change the suspension pH and evaluate internalization with a time resolution of a few seconds. We observe that internalized ligand can be detected within 1 min and that the rate is proportional to the number of receptors occupied. The rate is essentially linear over the first few minutes and approximately 60% of the receptor-bound ligand is internalized after 3 min.     

AMP and adenosine are both ligands for adenosine 2B receptor signaling

Adenosine is considered the canonical ligand for the adenosine 2B receptor (A_2BR). A_2BR is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A_2BR signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A_2BR.The computational modeling suggested that AMP interacts with more favorable energy to A_2BR compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A_2BR, indicating preferential signaling via the G_q pathway. Therefore, a putative AMP-A_2BR interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential G_q activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI.     

Capsaicin activation of the pain receptor,VR1: multiple open states from both partial and full binding

Capsaicin, the pungent ingredient of hot peppers, has long been used to identify nociceptors. Its molecular target, the vanilloid receptor VR1, was recently cloned and confirmed functionally as a polymodal detector of multiple pain stimuli: heat, acid, and vanilloids. Previous electrophysiology studies have focused on whole-cell characteristics of the receptor. Here, we provide the first in-depth single-channel kinetic study of VR1 to understand its activation mechanism. At low to medium concentrations, channel activity appeared as bursts. Not only did the durations of the interburst gaps vary with capsaicin, the bursts also appeared ligand-dependent, with high capsaicin prolonging bursts and stabilizing openings. Gating involved at least five closed and three open states, with strong correlations between short closures and long openings, and long closures and short openings. Increasing capsaicin reduced the long closures with little effect on short ones. The open time constants changed little with capsaicin concentration, though their relative proportions varied. These results suggest that 1), the channel contains multiple capsaicin binding sites; 2), both partial and full binding are capable of opening the channel; 3), when activated, multiple open states are accessible irrespective of the level of binding; and 4), capsaicin association occurs preferentially to the closed channel.     

T cell receptor binding transition states and recognition of peptide/MHC

T cell receptor recognition of peptide/MHC has been described as proceeding through a "two-step" process in which the TCR first contacts the MHC molecule prior to formation of the binding transition state using the germline-encoded CDR1 and CDR2 loops. The receptor then contacts the peptide using the hypervariable CDR3 loops as the transition state decays to the bound state. The model subdivides TCR binding into peptide-independent and peptide-dependent steps, demarcated at the binding transition state. Investigating the two-step model, here we show that two TCRs that recognize the same peptide/MHC bury very similar amounts of solvent-accessible surface area in their transition states. However, 1300-1500 A2 of surface area is buried in each, a significant amount suggestive of participation of peptide and associated CDR3 surface. Consistent with this interpretation, analysis of peptide and TCR variants indicates that stabilizing contacts to the peptide are formed within both transition states. These data are incompatible with the original two-step model, as are transition state models built using the principle of minimal frustration commonly employed in the investigation of protein folding and binding transition states. These findings will be useful in further explorations of the nature of TCR binding transition states, as well as ongoing efforts to understand the mechanisms by which T cell receptors recognize the composite peptide/MHC surface.