Impact of noise on self-rated job satisfaction and health in open-plan offices: a structural equation modelling approach.

This study uses a structural equation model to examine the effects of noise on self-rated job satisfaction and health in open-plan offices. A total of 334 employees from six open-plan offices in China and Korea completed a questionnaire survey. The questionnaire included questions assessing noise disturbances and speech privacy, as well as job satisfaction and health. The results indicated that noise disturbance affected self-rated health. Contrary to popular expectation, the relationship between noise disturbance and job satisfaction was not significant. Rather, job satisfaction and satisfaction with the environment were negatively correlated with lack of speech privacy. Speech privacy was found to be affected by noise sensitivity, and longer noise exposure led to decreased job satisfaction. There was also evidence that speech privacy was a stronger predictor of satisfaction with environment and job satisfaction for participants with high noise sensitivity. In addition, fit models for employees from China and Korea showed slight differences. PRACTITIONER SUMMARY: This study is motivated by strong evidence that noise is the key source of complaints in open-plan offices. Survey results indicate that self-rated job satisfaction of workers in open-plan offices was negatively affected by lack of speech privacy and duration of disturbing noise.

Medial epicanthoplasty without a vertical scar.

BACKGROUND: The epicanthal fold (Mongolian fold) in Asians reduces the aesthetic results of eyelid surgery, and thus, medial epicanthoplasty is commonly performed in combination with a double fold operation or blepharoptosis correction. Epicanthoplasty is one of the most popular cosmetic operations conducted in Asia, but scarring is a common problem. METHODS: From December of 2006 to July of 2011, we treated 60 cases using our epicanthoplasty method, which was designed to reduce scarring. A double fold operation and blepharoptosis correction was performed along with epicanthoplasty in 54 cases and an epicanthoplasty without a double fold operation in the remaining 6 cases. Follow-up periods ranged from 6 months to 4 years and 10 months. Previously, we used an elliptical excision epicanthoplasty method, which was simple and practical. However, the elliptical excision method leaves a vertical scar on the nasal side of the upper and lower eyelids. To avoid this scar, we placed an additional incision parallel with the ciliary margin of the lower and/or upper eyelids. The results of epicanthoplasty were evaluated by asking the patients and the surgeon involved to allocate visual analog scale scores. RESULTS: With the exception of 1 case of hypertrophic scarring and 4 cases of undercorrection, patients were satisfied with their results. Mean patient and surgeon visual analog scale scores were 4.6 and 4.2, respectively. The advantages of the described procedure are its simplicity and the minimal scarring caused in the epicanthal area. CONCLUSIONS: This method could become an effective means of removing the Asian epicanthal fold and minimizing vertical scars.

The possibility of microbial cellulose for dressing and scaffold materials.

In recent years, natural polymers such as cellulose, alginate and chitosan have been used worldwide as biomedical materials and devices, as they offer more advantages over synthetic polymers. The aim of this study was to clarify the usefulness of microbial cellulose (MC) for use as a dressing and scaffold material. For evaluating the biodegradability and toxicity of MC, we divided the rats (n = 12) into two groups (the implanted group and the non-implanted group). In the implanted group, we implanted the film type of MC in the backs of six rats. In the non-implanted group, however, we did not implant the film type of MC in the backs of the six rats. Four weeks later, we compared two groups by the gross, histological and biochemical characteristics by using blood and tissue samples. To evaluate the wound healing effects of MC, three full-thickness skin defects were made on the backs of each rat (n = 20). Three wounds on the backs of the same rats were treated with other dressing materials, namely, Vaseline gauze (group Con), Algisite M(®) (group Alg) and MC (group MC). We analysed the gross, histological and biochemical characteristics by western blotting. MC was found to be biodegradable and non-toxic. On day 3, the MC film was visible under the subcutaneous tissue; however, after 4 weeks, no remnants of the film were visible under the subcutaneous tissue. Furthermore, there was no evidence of MC-induced toxicity. Moreover, group MC showed more rapid wound healing compared with group Con. On day 14 after skin excision, group MC showed greater decrease in wound size compared with group Con (33% versus 7·2%). The wound healing effects were also substantiated by the histological findings (greater reduction in inflammation and rapid collagen deposition as well as neovascularisation) and western blotting (decreased expression of vascular endothelial growth factor and transforming growth factor-ß1 in group MC on day 14 after skin excision, unlike group Con). This study showed that, in addition to having wound healing effects, MC is biodegradable and non-toxic and can, therefore, be used as a dressing and scaffold material.

Inhibition of L-type amino acid transporter modulates the expression of cell cycle regulatory factors in KB oral cancer cells.

The purpose of this study was to examine the effect of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), an inhibitor of L-type amino acid transporters, on the cell growth suppression in KB human oral cancer cells and to study the roles of cell cycle regulatory factors in the BCH-induced growth inhibition. The effect of BCH on cell growth suppression and the influence of BCH to cell cycle regulatory factors in KB cell growth inhibition were examined using cell cycle analysis, immunoblotting and immunoprecipitation. The BCH treatment induced cell cycle arrest at G1 phase in KB cells. The expression of cyclin D3 was remarkably decreased by BCH treatment. The BCH inhibited the expression of cyclin-dependent protein kinase 6 (CDK6) in a time-dependent manner. In addition, the expression of CDK inhibitor p27 was increased by BCH treatment in KB cells, but not CDK inhibitors p21 and p15. These results suggest that, in KB cells, the inhibition of LAT1 by BCH causes cell cycle arrest at G1 phase by inhibiting cyclin D3-CDK6 complex whereas increasing expression of a CDK inhibitor p27.

Identification of specific transmembrane residues and ligand-induced interface changes involved in homo-dimer formation of a yeast G protein-coupled receptor.

The Saccharomyces cerevisiae alpha-factor pheromone receptor, Ste2p, has been studied as a model for G protein-coupled receptor (GPCR) structure and function. Dimerization has been demonstrated for many GPCRs, although the role(s) of dimerization in receptor function is disputed. Transmembrane domains one (TM1) and four (TM4) of Ste2p were shown previously to play a role in dimerization. In this study, single cysteine substitutions were introduced into a Cys-less Ste2p, and disulfide-mediated dimerization was assessed. Six residues in TM1 (L64 to M69) that had not been previously investigated and 19 residues in TM7 (T278 to A296) of which 15 were not previously investigated were mutated to create 25 single Cys-containing Ste2p molecules. Ste2p mutants V68C in TM1 and nine mutants in TM7 (cysteine substituted into residues 278, 285, 289, and 291 to 296) showed increased dimerization upon addition of an oxidizing agent in comparison to the background dimers formed by the Cys-less receptor. The formation of dimers was decreased for TM7 mutant receptors in the presence of alpha-factor indicating that ligand binding resulted in a conformational change that influenced dimerization. The effect of ligand on dimer formation suggests that dimers are formed in the resting state and the activated state of the receptor by different TM interactions.

Detergent-associated solution conformations of helical and beta-barrel membrane proteins.

Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the beta-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.

Evidence of the neuron-restrictive silencer factor (NRSF) interaction with Sp3 and its synergic repression to the mu opioid receptor (MOR) gene.

Previously, we reported that the neuron-restrictive silencer element (NRSE) of mu opioid receptor (MOR) functions as a critical regulator to repress the MOR transcription in specific neuronal cells, depending on neuron-restriction silence factor (NRSF) expression levels [C.S.Kim, C.K.Hwang, H.S.Choi, K.Y.Song, P.Y.Law, L.N. Wei and H.H.Loh (2004) J. Biol. Chem., 279, 46464-46473]. Herein, we identify a conserved GC sequence next to NRSE region in the mouse MOR gene. The inhibition of Sp family factors binding to this GC box by mithramycin A led to a significant increase in the endogenous MOR transcription. In the co-immunoprecipitation experiment, NRSF interacted with the full-length Sp3 factor, but not with Sp1 or two short Sp3 isoforms. The sequence specific and functional binding by Sp3 at this GC box was confirmed by in vitro gel-shift assays using either in vitro translated proteins or nuclear extract, and by in vivo chromatin immunoprecipitation assays. Transient transfection assays showed that Sp3-binding site of the MOR gene is a functionally synergic repressor element with NRSE in NS20Y cells, but not in the NRSF negative PC12 cells. The results suggest that the synergic interaction between NRSF and Sp3 is required to negatively regulate MOR gene transcription and that transcription of MOR gene would be governed by the context of available transcription factors rather than by a master regulator.

Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor type 1 membrane translocation and function.

UNLABELLED: PTHR1 mutants lacking endogenous cysteines in transmembrane and intracellular domains were generated. Mutant receptors were tested for their biological activities and mRNA and cell surface expression levels. C217 in intracellular loop 1 was determined to play a critical role in cell surface translocation and function of the receptor. INTRODUCTION: Elucidating the role of different domains of PTH receptor 1 (PTHR1) is essential for understanding the mechanism of ligand-receptor interactions. Here we present a study directed at determining the importance of cysteine residues present in the intracellular and transmembrane (TM) domains of the receptor. MATERIALS AND METHODS: Mutant receptors were generated by site-directed mutagenesis. Biological activities were characterized by adenylyl cyclase and competition binding assays. RT-PCR, ELISA, and immunofluorescence microscopy were carried out to determine receptor mRNA and protein expression levels. RESULTS: Mutations C460L and C462L in TM7, C568L in the C-terminal intracellular domain of the receptor, and removal of C397 in intracellular loop (ICL)3 by insertion of cleavage sites for Factor Xa did not affect binding affinity of PTH or agonist-induced adenylyl cyclase activity, although maximal responses (IC(max) and EC(max)) were decreased. However, mutations C217L in ICL1 or both C217L and C568L simultaneously resulted in a decrease in binding and loss of adenylyl cyclase activity. RT-PCR results showed that the observed changes in binding and activity were not caused by changes in mRNA expression. Next, we determined cell surface and total expression of the wildtype and mutant receptors by ELISA. We found that mutations of C460/C462 to L moderately decreased transfer of receptors to the cell surface. However, mutation of C217 to L in the ICL1 drastically reduced cell surface expression. Immunofluorescence and confocal microscopy studies confirmed reduced cell surface expression of receptors containing the C217L mutation. Similar results were obtained when replacing C217 and C460/C462 of the receptor with A instead of L. CONCLUSIONS: Our studies indicate that the cysteine at position 217 in ICL1 plays a critical role in translocation to the cell surface and biological function of PTHR1.

NRSF/REST regulates the mTOR signaling pathway in oral cancer cells.

The neuron-restrictive silencer factor/repressor element 1-silencing transcription factor (NRSF/REST) was originally discovered as a transcriptional repressor of neuronal genes in non-neuronal cells. However, it was recently reported to be abundantly expressed in several types of aggressive cancer cells, as well as in mature neurons. In the present study, the role of NRSF/REST in the human oral squamous cell carcinoma (SCC) KB cell line was evaluated. NRSF/REST was expressed at a higher level in KB cells when compared with that in normal human oral keratinocytes (NHOKs). Knockdown of NRSF/REST by siRNA reduced cell viability only in KB cells in a time-dependent manner, and this effect was due to the activation of apoptosis components and DNA fragmentation. In addition, knockdown of NRSF/REST disrupted the mTOR signaling pathway which is a key survival factor in many types of cancer cells. For example, the phosphorylation of elF4G, elF4E and 4E-BP1 was significantly reduced in the KΒ cells upon NRSF/REST knockdown. These results imply that NRSF/REST plays an important role in the survival of oral cancer cells by regulating the mTOR signaling pathway.

Transcriptional regulation of the neuropeptide VGF by the neuron-restrictive silencer factor/neuron-restrictive silencer element.

The neurotrophin-inducible gene VGF plays an important role in the maintenance of organismal energy balance and in the mediation of hippocampal synaptic activity. The regulatory mechanism of VGF transcription is not fully understood. The neuron-restrictive silencer factor (NRSF) binds with the neuron-restrictive silencer element (NRSE), thereby suppressing the transcription of NRSE-containing genes. In this study, we show that the NRSE sequence of the VGF gene critically regulates the repression of VGF expression in NMB cells. Sequence analysis also establishes the presence of two putative NRSEs (NRSE-1 and NRSE-2) in the promoter region of the VGF gene. In reporter gene experiments, a more than eight-fold increase in the promoter activity was observed when both NRSE-1 and NRSE-2 were deleted. Deletion of NRSE-2 alone did not affect the promoter activity, thus indicating that NRSE-1 could be solely responsible for the repression of VGF gene expression. Mutations in the NRSE-1 sequence increased promoter activity. However, no change in activity was observed when NRSE-1 was coexpressed with dominant-negative NRSF, thereby suggesting that endogenous NRSF interacts with NRSE-1. Binding of NRSF to NRSE in a sequence-specific manner was confirmed with chromatin immunoprecipitation assays, respectively. Furthermore, the overexpressed NRSF in PC12 cells significantly suppressed the VGF gene expression by interacting with the NRSE located in the VGF promoter region. Our results indicate that NRSF plays an important role as a repressor of VGF gene regulation in NMB cells through a mechanism that is dependent on VGF-NRSE.

Comparison of Blepharoptosis Correction Using Müller-aponeurosis Composite Flap Advancement and Frontalis Muscle Transfer.

BACKGROUND: Treatments for severe blepharoptosis are well documented and include the most common operations for restoring upper eyelid ptosis, which are levator surgery and frontal muscle transfers; however, the choice of treatment is still controversial. There are different approaches to the restoration of upper eyelid ptosis, and the choice will be based on ptosis severity and the surgeon's skill and experience. METHODS: Two hundred and fourteen patients presenting with a levator function of between 2 and 4 mm received ptosis correction between 1991 and 2010 at our clinic. Of these, 71 patients underwent Müller aponeurosis composite flap advancement for correction of 89 eyelids, and frontalis muscle transfer was performed on 143 patients (217 eyelids). Postoperative results were evaluated with an average follow-up period of 23 months. RESULTS: The preoperative average for marginal reflex distance (MRD1) in the Müller aponeurosis composite flap advancement group was 1.25 mm, and in the frontal muscle transfer group, it was 0.59 mm. The area of corneal exposure (ACE) was 57.2% in the Müller aponeurosis composite flap advancement group and 53.6% in the frontal muscle transfer group. The postoperative average distance was not significantly different for the 2 techniques. In the Müller aponeurosis composite flap advancement group, MRD1 was 2.7 mm and ACE was improved to 73.5%. In the frontal muscle transfer group, MRD1 was 2.3 mm and ACE was 71.2%. Undercorrection and eyelid asymmetry were the most frequently observed postoperative complications for both techniques. CONCLUSIONS: In our study, we confirmed that Müller aponeurosis composite flap advancement and the frontalis transfer technique are both effective in the correction of severe blepharoptosis; our results showed no significant differences between the 2 techniques.

A case of heel reconstruction with a reverse sural artery flap in a hemophilia B patient.

Hemophilia B is a rare blood coagulation disorder. Complications such as bleeding and hematoma can cause necrosis of flaps, wound disruption, and the disturbance of wound healing. In particular, guidelines for flap operations in hemophilia B patients have still not been defined, and case reports are rare. We reconstructed the heel of a 41-year-old male hemophilia B patient using a reverse sural artery flap operation. The patient presented with mild hemophilia, having 27% of the normal value of coagulation factor IX. Coagulation and the changing value of the coagulation factor were regularly measured, and 70% of the normal value of coagulation factor IX was maintained through the injection of recombinant coagulation factors and antihemorrhagics. Hematoma developed twice (postoperative day [POD] 5 and POD 7) and in each case the hematoma was removed. Injections of recombinant coagulation factors and antihemorrhagics were continuously administered until postoperative week 2. When the coagulation factors were within normal ranges. In this article, a hemophilia B patient underwent reverse sural artery flap surgery and the healing progress was analyzed. We conclude that higher than baseline levels of coagulation factors are needed for successful healing in reverse sural artery flap surgery.

Tubularized penile-flap urethroplasty using a fasciocutaneous random pedicled flap for recurrent anterior urethral stricture.

This report describes the use of a tubularized random flap for the curative treatment of recurrent anterior urethral stricture. Under the condition of pendulous lithotomy and suprapubic cystostomy, the urethral stricture was removed via a midline ventral penile incision followed by elevation of the flap and insertion of an 18-Fr catheter. Subcutaneous buried interrupted sutures were used to reapproximate the waterproof tubularized neourethra and to coapt with the neourethra and each stump of the urethra, first proximally and then distally. The defect of the penile shaft was covered by advancement of the surrounding scrotal flap. The indwelling catheter was maintained for 21 days. A 9 month postoperative cystoscopy showed no flap necrosis, no mechanical stricture, and no hair growth on the lumen of the neourethra. The patient showed no voiding discomfort 6 months after the operation. The advantages of this procedure are the lack of need for microsurgery, shortening of admission, the use of only spinal anesthesia (no general anesthesia), and a relatively short operative time. The tubularized unilateral penile fasciocutaneous flap should be considered an option for initial flap urethroplasty as a curative technique.

Spontaneous sinus conversion of permanent atrial fibrillation during treatment of hyperkalemia.

Hyperkalemia is a common adverse effect of treatment for heart failure and is associated with high mortality and morbidity. The cardiac manifestations of hyperkalemia include various electrocardiogram changes. We describe a case of a 74-year-old woman with heart failure and permanent atrial fibrillation who reverted to normal sinus rhythm during recovery from hyperkalemia.

Low-dose coronary computed tomography angiography using prospective ECG-triggering compared to invasive coronary angiography.

To assess the diagnostic accuracy of prospective ECG-triggering 64-slice multidetector computed tomography (MDCT) coronary angiography for evaluation of coronary artery disease (CAD). Forty-two patients (31 males, 11 females, mean age 64 years) underwent cardiac CT and invasive coronary angiography (ICA). Patients with a heart rate of <65 beats/min with stable heart rhythm were included in the study sample. We used a prospective ECG-triggering protocol. Luminal narrowing over 50% was considered to be significant according to a modified 17-segment AHA model, using invasive coronary angiography (ICA) as the standard of reference. The mean radiation dose was 3.5 mSv ± 0.3 (range, 3.3-4.2 mSv), and 542 of 549 segments (98.7%) in the 42 patients were diagnostic. In contrast, 119 of 542 segments (22%) were diagnosed as significant by ICA. The sensitivity, specificity, accuracy, PPV and NPV were 95.0, 96.2, 96, 85.8 and 98.8%, respectively. False positive results were affected by densely calcified plaques, whereas false negatives were caused by motion artifact with poor vessel attenuation at the distal segments or near the bifurcation area of the coronary arteries. Prospective ECG-triggering MDCT is a useful method for evaluating CAD in patients with a lower heart rate with low radiation dose.

The first extracellular loop of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p undergoes a conformational change upon ligand binding.

In this study of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p, we present data indicating that the first extracellular loop (EL1) of the alpha-factor receptor has tertiary structure that limits solvent accessibility and that its conformation changes in a ligand-dependent manner. The substituted cysteine accessibility method was used to probe the solvent exposure of single cysteine residues engineered to replace residues Tyr(101) through Gln(135) of EL1 in the presence and absence of the tridecapeptide alpha-factor and a receptor antagonist. Surprisingly, many residues, especially those at the N-terminal region, were not solvent-accessible, including residues of the binding-competent yet signal transduction-deficient mutants L102C, N105C, S108C, Y111C, and T114C. In striking contrast, two N-terminal residues, Y101C and Y106C, were readily solvent-accessible, but upon incubation with alpha-factor labeling was reduced, suggesting a pheromone-dependent conformational change limiting solvent accessibility had occurred. Labeling in the presence of the antagonist, which binds Ste2p but does not initiate signal transduction, did not significantly alter reactivity with the Y101C and Y106C receptors, suggesting that the alpha-factor-dependent decrease in solvent accessibility was not because of steric hindrance that prevented the labeling reagent access to these residues. Based on these and previous observations, we propose a model in which the N terminus of EL1 is structured such that parts of the loop are buried in a solvent-inaccessible environment interacting with the extracellular part of the transmembrane domain bundle. This study highlights the essential role of an extracellular loop in activation of a G protein-coupled receptor upon ligand binding.

Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p.

We present an example of expression and purification of a biologically active G-protein coupled receptor (GPCR) from yeast. An expression vector was constructed to encode the Saccharomyces cerevisiae GPCR alpha-factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Ligand binding and signaling assays of the epitope-tagged, mutated receptor showed it maintained the full wild-type biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5% n-dodecyl maltoside (DM). Approximately 120 microg of purified alpha-factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (K(d)) of the purified alpha-factor receptor in DM micelles was 28 nM as compared to K(d)=12.7 nM for Ste2p in cell membranes, and approximately 40% of the purified receptor was correctly folded as judged by ligand saturation binding. About 50% of the receptor sequence was retrieved from MALDI-TOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the alpha-factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

The mid-region of parathyroid hormone (1-34) serves as a functional docking domain in receptor activation.

Elucidating the bimolecular interface between parathyroid hormone (PTH) and its cognate G protein-coupled receptor (PTHR1) should yield insights into the basis of molecular recognition and the mechanism of ligand-mediated intracellular signaling for a system that is critically important in regulating calcium levels in blood. We used photoaffinity scanning (PAS) to identify key ligand-receptor interactions for residues from the unstructured mid-region domain of PTH-(1-34). Four PTH analogues, containing a single photoreactive p-benzoylphenylalanine (Bpa) residue in position 11, 15, 18, or 21, were found to photo-cross-link within receptor regions [165-176], [183-189], [190-298], and [165-176], respectively. Addition of these mid-region contacts as constraints to our previously proposed model of the PTH-PTHR1 complex and extensive molecular simulation experiments enables substantial refinement of the model. Specifically, (1) the overall receptor-bound conformation of the hormone is not extended, but bent; (2) helix [169-176] of the N-terminal extracellular domain (N-ECD) of the receptor is redirected toward the heptahelical bundle; and (3) the hormone traverses between the top of transmembrane (TM) helices 1 and 2, rather than between TM-7 and TM-1. This significantly alters the model of both the receptor-bound tertiary structure of the hormone and the topological orientation of the C-terminus of the N-ECD in the hormone-receptor bimolecular complex. We propose that the mid-region of PTH-(1-34) has a role in fixing, by extensive contacts with the receptor, the entry of the N-terminal helix of the hormone into the heptahelical bundle between TM-1 and TM-2. This anchorage would orient the amino terminus into position to activate the receptor.

Tyr266 in the sixth transmembrane domain of the yeast alpha-factor receptor plays key roles in receptor activation and ligand specificity.

To identify interactions between Ste2p, a G protein-coupled receptor of the yeast Saccharomyces cerevisiae, and its tridecapeptide ligand, alpha-factor (WHWLQLKPGQPMY), a variety of alpha-factor analogues were used in conjunction with site-directed mutagenesis of a targeted portion of Ste2p transmembrane domain six. Alanine substitution of residues in the 262-270 region of Ste2p did not affect pheromone binding or signal transduction, except for the Y266A mutant, which did not transduce signal yet exhibited only a small decrease in alpha-factor binding affinity. Substitutions with Ser, Leu, or Lys at Y266 also generated signaling-defective receptors. In contrast, Phe or Trp substitution at Y266 retained receptor function, suggesting that aromaticity at this position was critical. When coexpressed with WT receptor, the Y266A receptor exhibited a strong dominant-negative phenotype, indicating that this mutant bound G protein. A partial tryptic digest revealed that, in the presence of agonist, a different digestion profile for Y266A receptor was generated in comparison to that for WT receptor. The difference in trypsin-sensitive sites and their negative dominance indicated that the Y266A receptor was not able to switch into an "activated" conformation upon ligand binding. In comparison to WT Ste2p, the mutantY266A receptor showed increased binding affinity for N-terminal, alanine-substituted alpha-factor analogues (residues 1-4) and the antagonist [desW(1),desH(2)]alpha-factor. A substantial decrease in affinity was observed for alpha-factor analogues with Ala substitutions from residues 5-13. The results suggest that Y266 is part of the binding pocket that recognizes the N-terminal portion of alpha-factor and is involved in the transformation of Ste2p into an activated state upon agonist binding.