Endoscopic vacuum therapy for treatment of spontaneous and iatrogenic upper gastrointestinal defects

Background/Aims@#Endoscopic vacuum therapy (EVT) can heal a variety of defects within the gastrointestinal (GI) tract via applying negative pressure, which reduces the defect size, aspirates the infected fluid, and promotes granulation tissue. Here we present our experience with EVT as it relates to both spontaneous and iatrogenic upper GI tract perforations, leaks, and fistulas. @*Methods@#This retrospective study was conducted at four large hospital centers. All patients who underwent EVT between June 2018 and March 2021 were included. Data on multiple variables were collected, including demographics, defect size and location, number and intervals of EVT exchanges, technical success, and hospital length of stay. Student t-test and the chi-squared test were used to analyze the data. @*Results@#Twenty patients underwent EVT. The most common defect cause was spontaneous esophageal perforation (50%). The most common defect location was the distal esophagus (55%). The success rate was 80%. Seven patients were treated with EVT as the primary closure method. The mean number of exchanges was five with a mean interval of 4.3 days between exchanges. The mean length of hospital stay was 55.8 days. @*Conclusions@#EVT is a safe and effective initial management option for esophageal leaks and perforations.

Evaluating the Revised American Society for Gastrointestinal Endoscopy Guidelines for Common Bile Duct Stone Diagnosis

Background/Aims@#The American Society for Gastrointestinal Endoscopy (ASGE) revised its guidelines for risk stratification of patients with suspected choledocholithiasis. This study aimed to assess the diagnostic performance of the revision and to compare it to the previous guidelines. @*Methods@#We conducted a retrospective cohort study of 267 patients with suspected choledocholithiasis. We identified high-risk patients according to the original and revised guidelines and examined the diagnostic accuracy of both guidelines. We measured the association between individual criteria and choledocholithiasis. @*Results@#Under the original guidelines, 165 (62%) patients met the criteria for high risk, of whom 79% had confirmed choledocholithiasis. The categorization had a sensitivity and specificity of 68% and 55%, respectively, for the detection of choledocholithiasis. Under the revised guidelines, 86 (32%) patients met the criteria for high risk, of whom 83% had choledocholithiasis. The revised categorization had a lower sensitivity and higher specificity of 37% and 80%, respectively. The positive predictive value of the high-risk categorization increased with the revision, reflecting a potential decrease in diagnostic endoscopic retrograde cholangiopancreatograpies (ERCPs). Stone visualized on imaging had the greatest specificity for choledocholithiasis. Gallstone pancreatitis was not associated with the risk for choledocholithiasis. @*Conclusions@#The 2019 revision of the ASGE guidelines decreases the utilization of ERCP as a diagnostic modality and offers an improved risk stratification tool.

Evaluating the Revised American Society for Gastrointestinal Endoscopy Guidelines for Common Bile Duct Stone Diagnosis

Background/Aims@#The American Society for Gastrointestinal Endoscopy (ASGE) revised its guidelines for risk stratification of patients with suspected choledocholithiasis. This study aimed to assess the diagnostic performance of the revision and to compare it to the previous guidelines. @*Methods@#We conducted a retrospective cohort study of 267 patients with suspected choledocholithiasis. We identified high-risk patients according to the original and revised guidelines and examined the diagnostic accuracy of both guidelines. We measured the association between individual criteria and choledocholithiasis. @*Results@#Under the original guidelines, 165 (62%) patients met the criteria for high risk, of whom 79% had confirmed choledocholithiasis. The categorization had a sensitivity and specificity of 68% and 55%, respectively, for the detection of choledocholithiasis. Under the revised guidelines, 86 (32%) patients met the criteria for high risk, of whom 83% had choledocholithiasis. The revised categorization had a lower sensitivity and higher specificity of 37% and 80%, respectively. The positive predictive value of the high-risk categorization increased with the revision, reflecting a potential decrease in diagnostic endoscopic retrograde cholangiopancreatograpies (ERCPs). Stone visualized on imaging had the greatest specificity for choledocholithiasis. Gallstone pancreatitis was not associated with the risk for choledocholithiasis. @*Conclusions@#The 2019 revision of the ASGE guidelines decreases the utilization of ERCP as a diagnostic modality and offers an improved risk stratification tool.

The contribution of metaphor and metonymy to delusions.

This article investigates the possible role of metaphorical thinking in psychotic delusions. Twenty-five participants with delusions were asked to give an account of how their ideas had formed and to describe recent experiences relevant to their delusional beliefs. The data suggest that for some participants there may have been a crucial period when the person has unusual experiences, psychosocial difficulties, and made attempts involving metaphor/metonymy to understand these experiences. Furthermore, some participants reported very recent unusual experiences using metaphorical terms, and we speculate on the possibility that the content of the metaphors contributes to a continuation of psychotic experience. The data form a series of case illustrations and are exploratory. No generalizations can be made, but the presence of significant metaphors and metonymy in 11 out of 25 case histories suggests the process may be an important one. We end by outlining a theoretical model of how metaphors might contribute to the formation of delusions: it is suggested that delusional statements are intended to be literal statements, but report on experiences transformed by metaphorical meaning. This transformation involves the 'fusion' of conceptual domains.

Nonpeptidic, monocharged, cell permeable ligands for the p56lck SH2 domain.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

Correspondence between delusions and personal goals: a qualitative analysis.

OBJECTIVES: This pilot study describes a qualitative method for exploring delusions in terms of motivational themes. DESIGN: A semi-structured interview schedule was developed on the basis of an elementary conceptual frame specifying research questions. The analysis of each case uses a structured format. Triangulation was used to check: (i) reliability of motive categories; (ii) their consistent application to delusions. METHODS OF ANALYSIS: All patients had delusions and were diagnosed as having a psychotic disorder. Two types of analysis were used: (i) Interpretative phenomenological analysis with features of grounded analysis was used to classify motives. Data from 14 participants was used for this. (ii) The second phase was an examination of a possible correspondence of themes and involved: (a) a category-led thematic analysis of the delusion in terms of motivations; (b) a category-led thematic analysis of life goals and problems again in terms of motivations; and (c) an examination of correspondence between (a) and (b). RESULTS: The classification of goals and difficulties suggested six main categories: social connection; competence; experiential base (i.e. states of mind and body); material base (e.g. housing); direction; and evaluation (i.e. how a person evaluates himself or believes others evaluate him). Four cases are presented, each exploring the correspondence of themes. CONCLUSION: The methods of analysis seemed coherent and useful. In the cases presented, the delusions appeared to relate to fundamental concerns in a person's life.

Cloning and characterization of human Lnk, an adaptor protein with pleckstrin homology and Src homology 2 domains that can inhibit T cell activation.

Lnk was originally cloned from a rat lymph node cDNA library and shown to participate in T cell signaling. Human Lnk (hLnk) was cloned by screening a Jurkat cell cDNA library. hLnk has a calculated molecular mass of 63 kDa, and its deduced amino acid sequence indicates the presence of an N-terminal proline-rich region, a pleckstrin homology domain, and a Src homology 2 domain. When expressed in COS cells, hLnk migrates with an apparent molecular mass of 75 kDa. Confocal fluorescence microscope analysis indicates that in COS cells transfected with an expression vector encoding a chimeric Lnk-green fluorescent protein, hLnk is found at the juxtanuclear compartment and also appears to be localized at the plasma membrane. Lnk is tyrosine-phosphorylated by p56lck. Following phosphorylation, p56lck binds to tyrosine-phosphorylated hLnk through its Src homology 2 domain. In COS cells cotransfected with hLnk, p56lck, and CD8-zeta, hLnk associated with tyrosine-phosphorylated TCR zeta-chain through its Src homology 2 domain. The overexpression of Lnk in Jurkat cells led to an inhibition of anti-CD3 mediated NF-AT-Luc activation. Our study reveals a potentially new mechanism of T cell-negative regulation.

Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

Ligands for the tyrosine kinase p56lck SH2 domain: discovery of potent dipeptide derivatives with monocharged, nonhydrolyzable phosphate replacements.

p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.

Phosphotyrosine-containing dipeptides as high-affinity ligands for the p56lck SH2 domain.

Src homology-2 (SH2) domains are noncatalytic motifs containing approximately 100 amino acid residues that are involved in intracellular signal transduction. The phosphotyrosine-containing tetrapeptide Ac-pYEEI binds to the SH2 domain of p56lck (Lck) with an affinity of 0.1 microM. Starting from Ac-pYEEI, we have designed potent antagonists of the Lck SH2 domain which are reduced in peptidic character and in which the three carboxyl groups have been eliminated. The two C-terminal amino acids (EI) have been replaced by benzylamine derivatives and the pY + 1 glutamic acid has been substituted with leucine. The best C-terminal fragment identified, (S)-1-(4-isopropylphenyl)ethylamine, binds to the Lck SH2 domain better than the C-terminal dipeptide EI. Molecular modeling suggests that the substituents at the 4-position of the phenyl ring occupy the pY + 3 lipophilic pocket in the SH2 domain originally occupied by the isoleucine side chain. This new series of phosphotyrosine-containing dipeptides binds to the Lck SH2 domain with potencies comparable to that of tetrapeptide 1.

Effectiveness of cognitive therapy for delusions in routine clinical practice.

BACKGROUND: Previous studies have suggested that cognitive therapy is effective in modifying delusions. AIMS: To assess the effectiveness of cognitive therapy on patients seen in routine clinical work. METHOD: Eighteen patients with chronic delusions were treated using cognitive therapy, after the method of Chadwick and Lowe. A single-case multiple-baseline experimental design was used, including a control treatment. Each subject was used as their own control. RESULTS: Six patients reduced conviction in their delusions during cognitive therapy and not during the control treatment. Seven patients' conviction ratings did not change. Five patients showed a variable response. Degree of conviction did not fall to zero in any patient. All patients reported that the therapy had been helpful; six spontaneously mentioned changes in psychotic thinking. CONCLUSIONS: One-third of patients with chronic delusions whom we treated responded to delusion modification with a reduction in degree of belief. Change within therapy sessions predicted outcome, as did variation in the conviction during baseline. Cognitive therapy with delusions should aim at reducing distress as well as conviction.

Recombinant IkappaB kinases alpha and beta are direct kinases of Ikappa Balpha.

Activation of the transcription factor NF-kappaB is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IkappaB. A large multiprotein complex, the IkappaB kinase (IKK), catalyzes the phosphorylation of IkappaB. The two kinase components of the IKK complex, IKKalpha and IKKbeta, were overexpressed in insect cells and purified to homogeneity. Both purified IKKalpha and IKKbeta specifically catalyzed the phosphorylation of the regulatory serine residues of Ikappa Balpha. Hence, IKKalpha and IKKbeta were functional catalytic subunits of the IKK complex. Purified IKKalpha and IKKbeta also preferentially phosphorylated serine as opposed to threonine residues of Ikappa Balpha, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKKalpha and IKKbeta revealed that the kinase activity of IKKbeta on Ikappa Balpha is 50-60-fold higher than that of IKKalpha. The primary difference between the two activities is the Km for Ikappa Balpha. The kinetics of both IKKalpha and IKKbeta followed a sequential Bi Bi mechanism. No synergistic effects on Ikappa Balpha phosphorylation were detected between IKKalpha and IKKbeta. Thus, in vitro, IKKalpha and IKKbeta are two independent kinases of Ikappa Balpha.

Carboxymethyl-phenylalanine as a replacement for phosphotyrosine in SH2 domain binding.

The crystal structure of human p56(lck) SH2 domain in complex with an inhibitor containing the singly charged p-(carboxymethyl)phenylalanine residue (cmF) as a phosphotyrosine (Tyr(P) or pY) replacement has been determined at 1.8 A resolution. The binding mode of the acetyl-cmF-Glu-Glu-Ile (cmFEEI) inhibitor is very similar to that of the pYEEI inhibitor, confirming that the cmFEEI inhibitor has a similar mechanism of SH2 domain inhibition despite its significantly reduced potency. Observed conformational differences in the side chain of the cmF residue can be interpreted in terms of maintaining similar interactions with the SH2 domain as the Tyr(P) residue. The crystal structure of the free p56(lck) SH2 domain has been determined at 1.9 A resolution and shows an open conformation for the BC loop and an open phosphotyrosine binding pocket, in contrast to earlier studies on the src SH2 domain that showed mostly closed conformation. The structural information presented here suggests that the carboxymethyl-phenylalanine residue may be a viable Tyr(P) replacement and represents an attractive starting point for the design and development of SH2 domain inhibitors with better pharmaceutical profiles.

Interaction between the SH2 domains of ZAP-70 and the tyrosine-based activation motif 1 sequence of the zeta subunit of the T-cell receptor.

One of the key steps involved in T-cell activation is binding of the tyrosine kinase ZAP-70 via its two SH2 domains to peptide segments termed tyrosine-based activation motifs (ITAM) which are present in three of the T-cell receptor (TCR) subunits. The crystal structure of the ZAP-70 SH2 domains complexed to phosphopeptide revealed that the amino-terminal phosphotyrosine-binding pocket is formed at the interface between the two SH2 domains. This study was designed to further characterize the binding between TCR zeta ITAM1 and the ZAP-70 SH2 domains as well as to assess the change in conformation of SH2 domain structure upon zeta ITAM1 binding. BIAcore analysis of wild type and nonfunctional single-point mutants of ZAP-70 SH2 domains demonstrated that the amino-terminal SH2 domain can bind phosphopeptide in the absence of a functional carboxyl-terminal SH2 domain. In addition, the amino-terminal SH2 domain prefers the RREEpYDVLDK sequence of zeta chain ITAM1 over the GQNQLpYNELNL sequence. To assess changes in protein conformation upon ITAM binding to ZAP-70 SH2 domains, fluorescence spectroscopy and analytical ultracentrifugation experiments were performed. A significant blue shift in the tryptophan emission spectrum of the SH2 domains was observed in the presence of saturating amounts of phosphopeptide, indicating a loss in solvent exposure for the tryptophan residues in the protein-phosphopeptide complex. This was accompanied by changes in the frictional coefficient consistent with a compacting of the protein structure. Finally, thermal denaturation experiments showed an increase in stability and cooperativity in unfolding for the protein-phosphopeptide complex relative to the protein alone.

Binding affinities of the SH2 domains of ZAP-70, p56lck and Shc to the zeta chain ITAMs of the T-cell receptor determined by surface plasmon resonance.

The zeta chains of the T cell receptor complex play a critical role in the initiation of proximal signaling events upon T cell activation. Three pairs of potential tyrosine phosphorylation sites are located within the cytoplasmic domains of the zeta chains. Subsequent to engagement of the T cell receptor, one or more of these tyrosine residues is phosphorylated. The phosphotyrosine residues, along with flanking amino acids, form an activation motif (and are shared by signaling subunits in the TCR, B cell receptor, and FcgammaRI) termed tyrosine-based activation motifs (ITAMs). ITAMs serve as binding sites for SH2 domain-containing proteins. Recent evidence suggests that the zeta chains provide docking space for several key signal transduction molecules such as ZAP-70, p56lck, and Shc. To determine if ZAP-70, p56lck, and Shc bind to particular zeta chain ITAM sequences, quantitative free-solution measurements of binding affinities (Kd) were obtained by use of surface plasmon resonance technology. The results indicate that binding affinities of distinct SH2 domains to individual and paired phosphorylation sites greatly differ, and may dictate the sequence of signal transduction events.

Crystal structures of the human p56lck SH2 domain in complex with two short phosphotyrosyl peptides at 1.0 A and 1.8 A resolution.

src homology 2 (SH2) domains are modules of about 100 amino acid residues and bind to phosphotyrosine-containing motifs in a sequence-specific manner. They play important roles in intracellular signal transduction and represent potential targets for pharmacological intervention. The protein tyrosine kinase p56lck is a member of the src family and is involved in T-cell activation. The crystal structure of its SH2 domain with an 11-residue peptide showed that the phosphotyrosine and the Ile residue at the pY + 3 position are recognized by the SH2 domain. We present here the crystal structure of the SH2 domain of human p56lck in complex with the short phosphotyrosyl peptide Ac-pTyr-Glu-Glu-Ile (pYEEI peptide) at 1.0 A resolution. The structural analysis at atomic resolution reveals that residue Arg134 (alphaA2), which interacts with the phosphotyrosine side-chain, is present in two conformations in the complex. The structure at 1.8 A resolution of the complex with the phosphotyrosyl peptide Ac-pTyr-Glu-Glu-Gly (pYEEG peptide), which is 11 fold less potent, shows another binding mode for the pY + 3 residue as well as rearrangements of the side-chain of Arg196 (EF3) and one of the water molecules at the base of the pY + 3 pocket. The structure of the complex with the short pYEEI peptide at atomic resolution represents a good starting point for the design and optimization of new inhibitors. Comparative structural analysis of many different inhibitor complexes will be an important component of this drug discovery process.

Determination of receptor-ligand kinetic and equilibrium binding constants using surface plasmon resonance: application to the lck SH2 domain and phosphotyrosyl peptides.

Experimental and computational methods were developed for surface plasmon resonance (SPR) measurements involving interactions between a solution-binding component and a surface-immobilized ligand. These protocols were used to distinguish differences in affinity between the SH2 domain of lck and phosphotyrosyl peptides. The surface-immobilized ligand was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consensus sequence (pYEEI) for binding lck SH2. In the kinetic experiment, SPR phenomena were measured during association and dissociation reactions for a series of glutathione-S-transferase (GST)-SH2 concentrations, generating a set of SPR curves. A global computational analysis using an A + B<==>AB model resulted in single set of parameter estimates and statistics. In an abbreviated format, an equilibrium experiment was designed so that equilibrium constants (Keq) could be determined rapidly and accurately. A competitive equilibrium assay was developed for GST-SH2 in which Keq values for a series of phosphotyrosyl peptides (derived from the pYEEI sequence) varied over 3 orders of magnitude. Interestingly, these results highlighted the significance of the +1 glutamate in providing high-affinity binding to the SH2 domain. For most drug discovery programs, these Keq determinations are a sufficient measure of potency for the primary screen, with koff and kon determined in a secondary assay. Thus, the application of these techniques to SPR binding phenomena should prove valuable in the discovery and design of receptor-ligand antagonists.

Casein kinase II stimulates Xenopus laevis DNA topoisomerase I by physical association.

A Xenopus laevis casein kinase II-like activity copurified with X. laevis DNA topoisomerase I activity during chromatography on DEAE-cellulose, phosphocellulose, and hydroxylapatite, but the two activities were resolved by chromatography on DNA-agarose [Kaiserman, H. B., Ingebritsen, T. S., & Benbow, R. M. (1988) Biochemistry 27, 3216-3222]. Phosphorylation of the catalytic polypeptides of dephosphorylated X. laevis DNA topoisomerase I by the endogenous X. laevis casein kinase II-like activity apparently resulted in a severalfold increase in catalytic activity. In this study, we show that incubation of purified X. laevis DNA topoisomerase I with electrophoretically homogeneous bovine brain casein kinase II and ATP strongly stimulated catalytic activity. Surprisingly, purified bovine casein kinase II stimulated X. laevis DNA topoisomerase I activity by more than an order of magnitude in the absence of ATP, although ATP resulted in additional stimulation. Other basic proteins, such as histone H1 and HMG proteins, also stimulated X. laevis DNA topoisomerase I catalytic activity 2-3-fold in the absence of ATP. Modulation of catalytic activity by direct physical association (protein-protein interactions) must, therefore, be considered in addition to phosphorylation in assessing the physiological role of casein kinase II and other basic proteins during regulation of X. laevis DNA topoisomerase I activity in vivo.

Acidic residues are involved in substrate recognition by two soluble protein tyrosine phosphatases, PTP-5 and rrbPTP-1.

The mechanisms for substrate recognition by two cytoplasmic protein tyrosine phosphatases, PTP-5 and rrbPTP-1, were investigated. Phosphorylation sites on tyrosine-phosphorylated casein, a model PTP substrate, were characterized. Two peptides based on casein phosphorylation sites and one peptide based on the tyrosine phosphorylation site of reduced, carboxamidomethylated and maleylated (RCM) lysozyme were tested as PTP substrates. The three peptides were dephosphorylated by PTP-5 and rrbPTP-1 at rates comparable to those of the corresponding sites on the intact proteins. This indicates that peptides based on the two model PTP substrates, casein and RCM-lysozyme, contained all or most of the structural information necessary for PTP-5 and rrbPTP-1 substrate recognition. Structural elements required for substrate recognition by PTP-5 and rrbPTP-1 were also investigated. Km values for dephosphorylation of three simple aromatic phosphate esters (phosphotyrosine, p-nitrophenyl phosphate, and phenyl phosphate) by rrbPTP-1 were about 5000-fold higher than those obtained for the peptide and protein substrates. This indicates that recognition of protein and peptide substrates involves structural elements in addition to the phosphate group and the aromatic tyrosine ring of phosphotyrosine. Analysis of the effects of truncations and Ala for polar substitutions on the reactivity with PTP-5 and rrbPTP-1 of peptides based on casein, RCM-lysozyme, and angiotensin II indicated that Asp or Glu within the first five residues on the N-terminal side of phosphotyrosine increased peptide reactivity with both PTP's. Asn residues were unable or only weakly able to substitute for Asp residues.(ABSTRACT TRUNCATED AT 250 WORDS)

The cytoplasmic domain of the integrin lymphocyte function-associated antigen 1 beta subunit: sites required for binding to intercellular adhesion molecule 1 and the phorbol ester-stimulated phosphorylation site.

We have defined the regions of the cytoplasmic domain of the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) that are required for active binding of its extracellular domain to intercellular adhesion molecule 1 (ICAM-1). The NH2-terminal 28 amino acids in the cytoplasmic domain are dispensable, but a segment of 5 amino acids including three contiguous threonines (758-760) and Phe 766 in the COOH-terminal third of the cytoplasmic domain are required for binding to ICAM-1. Mutation and phosphoamino acid analysis show that Ser 756 is the major residue phosphorylated in response to phorbol ester. Furthermore, multiple mutations demonstrate that serine phosphorylation can be dissociated from phorbol ester-stimulated binding of LFA-1 to ICAM-1. The sites we have defined are previously unremarked, are well conserved in the beta 1, beta 3, and beta 7 integrin subunits, and may be of broad importance in regulating adhesiveness of integrins.