Proximal Signaling Molecules Research Articles

Regression of abdominal aortic aneurysm by inhibition of c-Jun N-terminal kinase

Abdominal aortic aneurysm (AAA) is a common disease among elderly people that, when surgical treatment is inapplicable, results in progressive expansion and rupture of the aorta with high mortality. Although nonsurgical treatment for AAA is much awaited, few options are available because its molecular pathogenesis remains elusive. Here, we identify JNK as a proximal signaling molecule in the pathogenesis of AAA. Human AAA tissue showed a high level of phosphorylated JNK. We show that JNK programs a gene expression pattern in different cell types that cooperatively enhances the degradation of the extracellular matrix while suppressing biosynthetic enzymes of the extracellular matrix. Selective inhibition of JNK in vivo not only prevented the development of AAA but also caused regression of established AAA in two mouse models. Thus, JNK promotes abnormal extracellular matrix metabolism in the tissue of AAA and may represent a therapeutic target.

Identification of a Molecular Therapeutic Target for Abdominal Aortic Aneurysm

Although we have gained much knowledge about the signal transduction pathways at the molecular level in physiological and pathophysiological conditions in the last decades, it is still a major challenge to identify a molecular therapeutic target in a given disease. We describe a signaling pathway-oriented global transcriptional analysis, which identified JNK, a stress-activated protein kinase, as a proximal signaling molecule in the pathogenesis of abdominal aortic aneurysm (AAA). AAA is a common disease among elderly people that, when surgical treatment is inapplicable, results in progressive expansion and rupture of the aorta with high mortality.

RIAM, a Novel Rap-1 Effector, Is Recruited at the Immunological Synapse and the Lipid Rafts and Links TCR-Mediated Signals to the Actin Cytoskeleton.

T cell receptor (TCR) ligation induces rapid polarization of the actin cytoskeleton resulting in the formation and stabilization of the immunological synapse (IS), recruitment of signaling molecules, and initiation of signaling cascades of T cell activation. Actin remodeling is essential for these events and is mandatory for T cell activation. Specific recruitment, redistribution and organization of signaling molecules in the IS is facilitated by lipid raft microdomains, which provide a scafold for focal protein assembly. Fyn and ZAP-70 are the most proximal TCR signaling molecules that localized in the IS and are redistributed in the lipid rafts during T cell activation. Currently, it is poorly understood how signals originating from the activated TCR are linked to the cytoskeletal rearrangement and the focal redistribution of signaling proteins. Recently, we identified RIAM, a novel adaptor molecule that contains a RA (Ras Association) domain, a PH (Plekstrin Homology) domain and proline-rich motifs. RIAM interacts specifically with active GTP-bound Rap1 and with regulators of the actin cytoskeleton Evl, VASP and Profilin. Profilin associates with G-actin and promotes actin polymerization by adding actin monomers to the barbed ends of F-actin. Ena/VASP family proteins are cytoskeletal proteins and regulate actin dynamics. Via these interactions, RIAM functions as a regulator of the actin cytoskeleton. To examine whether RIAM plays a role in the local reorganization of the actin cytoskeleton during IS formation, stable GFP-transfected Jurkat T cells were incubated with Raji B cells as APC, in the presence or absence of SEE. T cell:APC conjugates and formation of IS were examined by confocal microscopy. Synapse formation between GFP-Jurkat cells and Raji B cells was detected only in the presence of SEE. Under these conditions, polymerized (F) actin was highly recruited at the IS, as determined by phalloidin staining. Staining with RIAM-specific antibody indicated that in the absence of antigen, RIAM was diffusely expressed in the cytoplasm and at the plasma membrane. Impressively, upon incubation with SEE loaded Raji cells, RIAM was redistributed at the IS where it co-localized with polymerized (F) actin. Staining with ZAP-70 specific antibody revealed that ZAP-70 was also recruited at the IS upon activation and co-localized with RIAM and polymerized (F) actin. Sucrose gradient centrifugation indicated that RIAM translocated to the lipid rafts and co-localized in the same raft fraction with fyn and ZAP-70. RIAM was also a substrate of fyn and ZAP-70, which induced its tyrosine phosphorylation. Co-expression studies, by transfection of RIAM with fyn or ZAP-70 cDNA in COS cells, indicated that RIAM was a specific substrate of fyn and ZAP-70 but not lck. In vivo association of endogenous RIAM with fyn and ZAP-70 was also observed in primary T cells and in RIAM-transfected Jurkat cells and this interaction was highly upregulated upon activation. In the same complex, RIAM associated with grb2, crkL and the cytoskeletal proteins VASP and Profilin, indicating that RIAM functions as a link between the actin cytoskeleton and TCR signaling. Taken together these observations indicate a unique role of RIAM as an adaptor protein integrating TCR-mediated events that result in focal redistribution of polymerized actin, recruitment of signaling molecules at the IS and initiation of downstream signaling pathways.

Resistance to Fas-mediated apoptosis is restored by cycloheximide through the downregulation of cellular FLIPL in NK/T-cell lymphoma

Extranodal NK/T-cell lymphoma (NKTL), nasal type, is a highly aggressive neoplasm and is strongly associated with Epstein–Barr virus (EBV). In this study, we demonstrate that EBV-positive NKTL cell lines, namely, Hank-1, NK-YS, and NK-L, are resistant to Fas-mediated apoptosis induced by anti-Fas antibodies despite high levels of Fas surface expression and no mutation in the Fas gene. Fas stimulation of Hank-1 and NK-YS cells showed little processing of caspase 8, caspase 3, or bid, although the proximal signaling molecules of the death-inducing signaling complex, namely, Fas, Fas-associated protein with a death domain, caspase 8, and bid were present in these cells. Consistent with previous reports on the hypermethylation of death associated protein (DAP) kinase in NKTLs, the promoter of DAP kinase was methylated and its mRNA not detected in Hank-1 cells. However, the restoration of DAP kinase expression by 5-aza-2′-deoxycytidine did not sensitize Hank-1 to Fas-mediated apoptosis, indicating that DAP kinase deficiency does not contribute to resistance to Fas-mediated apoptosis. Since etoposide-induced apoptosis involved caspase 3 activation in Hank-1 and NK-YS cells, the caspase 3-dependent apoptotic machinery appears to be intact. Interestingly, cotreatment of Hank-1 with cycloheximide, a protein synthesis inhibitor, markedly sensitized cells to Fas-mediated apoptosis along with caspase 8 activation and c-FLIPL (cellular FLICE inhibitory protein long form) downregulation. Moreover, immunohistochemistry on paraffin-embedded tissue revealed c-FLIP expression in 39% (14 of 36) of NKTL patients. Taken together, these findings indicate that c-FLIPL-mediated resistance to Fas contributes to the development and progression of NKTLs. This study also suggests that agents capable of downregulating c-FLIPL could be used to treat NKTL.

Selective Inhibition of FcεRI-Mediated Mast Cell Activation by a Truncated Variant of Cbl-b Related to the Rat Model of Type 1 Diabetes Mellitus

Ubiquitin-protein ligase Cbl-b negatively regulates high affinity IgE receptor (FcepsilonRI)-mediated degranulation and cytokine gene transcription in mast cells. In this study, we have examined the role of a truncated variant of Cbl-b related to the rat model of type 1 diabetes mellitus using the mast cell signaling model. Overexpression of the truncated Cbl-b that lacks the C-terminal region did not suppress the activation of proximal and distal signaling molecules leading to degranulation. FcepsilonRI-mediated tyrosine phosphorylation of Syk, Gab2, and phospholipase C-gamma1, and activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAP kinase), and inhibitor of nuclear factor kappaB kinase (IKK), and generation of Rac1 are unaffected in cells overexpressing the truncated Cbl-b in the lipid raft. On the other hand, FcepsilonRI-mediated transcriptional activation of nuclear factor of activated T cells (NFAT), and transcription of interleukin-3 (IL-3) and IL-4 mRNA are inhibited by overexpression of the truncated variant of Cbl-b. This suppression parallels the re-compartmentalization of specific effector molecules in the lipid raft. These structural and functional analyses reveal the mechanism underlying the selective inhibition of cellular signaling by the truncated variant of Cbl-b related to insulin-dependent diabetes mellitus.

Proximal B cell receptor signaling pathways

AbstractEngagement of the B cell antigen receptor by antigen initiates a complex and interconnected cascade of signaling pathways that determine whether a B cell will divide, differentiate, or die. Both biochemical and genetic studies have defined the principal molecules, including the BCR components Igσ and Igβ, Src kinases, Syk, and Btk. Linker proteins such as BLNK have recently been shown to play a vital role in organizing proximal signaling molecules and coupling the BCR to distal signaling pathways. In this review, we will pay particular attention to how BCR‐proximal kinases coordinate the activation of PLCγ2, leading to the initiation and amplification of BCR‐mediated calcium flux and the activation of PI‐3 kinase.

Ribosomal S6 kinase as a mediator of keratinocyte growth factor-induced activation of Akt in epithelial cells.

The keratinocyte growth factor receptor (KGFR) is a member of the fibroblast growth factor receptor (FGFR) superfamily. The proximal signaling molecules of FGFRs are much less characterized compared with other growth factor receptors. Using the yeast two-hybrid assay, we have identified ribosomal S6 kinase (RSK) to be a protein that associates with the cytoplasmic domain of the KGFR. The RSK family of kinases controls multiple cellular processes, and our studies for the first time show association between the KGFR and RSK. Using a lung-specific inducible transgenic system we have recently demonstrated protective effects of KGF on the lung epithelium and have demonstrated KGF-induced activation of the prosurvival Akt pathway both in vivo and in vitro. Here we show that a kinase inactive RSK mutant blocks KGF-induced Akt activation and KGF-mediated inhibition of caspase 3 activation in epithelial cells subjected to oxidative stress. It was recently shown that RSK2 recruits PDK1, the kinase responsible for both Akt and RSK activation. When viewed collectively, it appears that the association between the KGFR and RSK plays an important role in KGF-induced Akt activation and consequently in the protective effects of KGF on epithelial cells.

Obligatory role for cooperative signaling by pre-TCR and Notch during thymocyte differentiation.

The first checkpoint during T cell development, known as beta selection, requires the successful rearrangement of the TCR-beta gene locus. Notch signaling has been implicated in various stages during T lymphopoiesis. However, it is unclear whether Notch receptor-ligand interactions are necessary during beta selection. Here, we show that pre-TCR signaling concurrent with Notch receptor and Delta-like-1 ligand interactions are required for the survival, proliferation, and differentiation of mouse CD4(-)CD8(-) thymocytes to the CD4(+)CD8(+) stage. Furthermore, we address the minimal signaling requirements underlying beta selection and show a hierarchical positioning of key proximal signaling molecules. Collectively, our results demonstrate an essential role for Notch receptor-ligand interactions in enabling the autonomous signaling capacity of the pre-TCR complex.

Characterization of a Novel and Functional Human Prolactin Receptor Isoform (ΔS1PRLr) Containing Only One Extracellular Fibronectin-Like Domain

Prolactin (PRL)-dependent signaling occurs as the result of ligand-induced homodimerization of the PRL receptor (PRLr). To date, short, intermediate, and long human PRLr isoforms have been characterized. To investigate the expression of other possible human PRLr isoforms, RT-PCR was performed on mRNA isolated from the breast carcinoma cell line T47D. A 1.5-kb PCR fragment was isolated, subcloned, and sequenced. The PCR product exhibited a nucleotide sequence 100% homologous to the human long isoform except bp 71-373 were deleted, which code for the S1 motif of the extracellular domain. Therefore, this isoform was designated the deltaS1 PRLr. Northern analysis revealed variable deltaS1 PRLr mRNA expression in a variety of tissues. Transfection of Chinese hamster ovary cells with deltaS1 cDNA showed the isoform is expressed at the protein level on the cell surface with a molecular mass of approximately 70 kDa. Kinetic studies indicated the deltaS1 isoform bound ligand at a lower affinity than wild-type receptor. The deltaS1 PRLr was also shown to activate the proximal signaling molecule Jak2 upon addition of ligand to transfected cells, and, unlike the long PRLr, high concentrations of ligand did not function as a self-antagonist to signaling during intervals of PRL serum elevation, i.e. stress and pregnancy. Given its apparent widespread expression, this PRLr isoform may contribute to PRL action. Furthermore, the functionality of this receptor raises interesting questions regarding the minimal extracellular domain necessary for ligand-induced receptor signaling.

Divergence of Bacterial Lipopolysaccharide Pro-apoptotic Signaling Downstream of IRAK-1

The vascular endothelium is a key target of circulating bacterial lipopolysaccharide (LPS). LPS elicits a wide array of endothelial responses, including the up-regulation of cytokines, adhesion molecules, and tissue factor, many of which are dependent on NF-kappa B activation. In addition, LPS has been demonstrated to induce endothelial apoptosis both in vitro and in vivo. Although the mechanism by which LPS activates NF-kappa B has been well elucidated, the signaling pathway(s) involved in LPS-induced apoptosis remains unknown. Using a variety of dominant negative constructs, we have identified a role for MyD88 and interleukin-1 receptor-associated kinase-1 (IRAK-1) in mediating LPS pro-apoptotic signaling in human endothelial cells. We also demonstrate that LPS-induced endothelial NF-kappa B activation and apoptosis occur independent of one another. Together, these data suggest that the proximal signaling molecules involved in LPS-induced NF-kappa B activation have a requisite involvement in LPS-induced apoptosis and that the pathways leading to NF-kappa B activation and apoptosis diverge downstream of IRAK-1.

Cellular Differentiation Causes a Selective Down-regulation of Interleukin (IL)-1β-mediated NF-κB Activation and IL-8 Gene Expression in Intestinal Epithelial Cells

Interleukin (IL)-1beta signals through various adapter proteins and kinases that lead to activation of numerous downstream targets, including the transcription factors including NF-kappaB. In this study, we analyzed and characterized the effect of the differentiation of intestinal epithelial cells on IL-1beta-mediated NF-kappaB activation and IL-8 gene expression. We report that IL-8 mRNA accumulation and protein secretion were down-regulated in IL-1beta- and lipopolysaccharide-stimulated differentiated HT-29 cells (HT-29/MTX, where MTX is methotrexate) compared with undifferentiated cells (HT-29/p), whereas no differential effects were found following tumor necrosis factor (TNF)-alpha or phorbol myristate acetate stimulation. Cross-linking and affinity binding studies reveal that IL-1beta exclusively binds the type I receptor (IL-1RI) and not IL-1RII in both HT-29/p and HT-29/MTX cells. IL-1beta-mediated IkappaB kinase and c-Jun N-terminal kinase (JNK) activity were both diminished in differentiated HT-29 cells. DNA binding activity in differentiated HT-29 cells relative to HT-29/p cells was strongly reduced following IL-1beta exposure but not after TNF-alpha stimulation. The proximal IL-1 signaling molecule IL-1 receptor-associated kinase was not degraded in IL-1beta-stimulated HT-29 cells, in contrast to Caco-2 cells. kappaB-luciferase reporter gene activity was 16-fold higher following TNF receptor-associated factor-6 transfection after IL-1beta stimulation in HT-29/MTX cells. We conclude that cellular differentiation of HT-29 cells selectively impairs the IL-1beta signaling pathway inhibiting both NF-kappaB and JNK activity in response to IL-1beta. This relative unresponsiveness to IL-1beta may represent an important regulatory mechanism of differentiated intestinal epithelial cells.

Insulin signaling and insulin sensitivity after exercise in human skeletal muscle.

Muscle glucose uptake, glycogen synthase activity, and insulin signaling were investigated in response to a physiological hyperinsulinemic (600 pmol/l)-euglycemic clamp in young healthy subjects. Four hours before the clamp, the subjects performed one-legged exercise for 1 h. In the exercised leg, insulin more rapidly activated glucose uptake (half activation time [t1/2] = 11 vs. 34 min) and glycogen synthase activity (t1/2 = 8 vs. 17 min), and the magnitude of increase was two- to fourfold higher compared with the rested leg. However, prior exercise did not result in a greater or more rapid increase in insulin-induced receptor tyrosine kinase (IRTK) activity (t1/2 = 50 min), serine phosphorylation of Akt (t1/2 = 1-2 min), or serine phosphorylation of glycogen synthase kinase-3 (GSK-3) (t1/2 = 1-2 min) or in a larger or more rapid decrease in GSK-3 activity (t1/2 = 3-8 min). Thirty minutes after cessation of insulin infusion, glucose uptake, glycogen synthase activity, and signaling events were partially reversed in both the rested and the exercised leg. We conclude the following: 1) physiological hyperinsulinemia induces sustained activation of insulin-signaling molecules in human skeletal muscle; 2) the more distal insulin-signaling components (Akt, GSK-3) are activated much more rapidly than the proximal signaling molecules (IRTK as well as insulin receptor substrate 1 and phosphatidylinositol 3-kinase [Wojtaszewski et al., Diabetes 46:1775-1781, 1997]); and 3) prior exercise increases insulin stimulation of both glucose uptake and glycogen synthase activity in the absence of an upregulation of signaling events in human skeletal muscle.

Signaling through sphingolipid microdomains of the plasma membrane: the concept of signaling platform.

Transmembrane signaling requires modular interactions between signaling proteins, phosphorylation or dephosphorylation of the interacting protein partners and temporary elaboration of supramolecular structures, to convey the molecular information from the cell surface to the nucleus. Such signaling complexes at the plasma membrane are instrumental in translating the extracellular cues into intracellular signals for gene activation. In the most straightforward case, ligand binding promotes homodimerization of the transmembrane receptor which facilitates modular interactions between the receptor's cytoplasmic domains and intracellular signaling and adaptor proteins. For example, most growth factor receptors contain a cytoplasmic protein tyrosine kinase (PTK) domain and ligand-mediated receptor dimerization leads to cross phosphorylation of tyrosines in the receptor's cytoplasmic domains, an event that initiates the signaling cascade. In other signaling pathways where the receptors have no intrinsic kinase activity, intracellular nonreceptor PTKs (i.e. Src family PTKs, JAKs) are recruited to the cytoplasmic domain of the engaged receptor. Execution of these initial phosphorylations and their translation into efficient cellular stimulation requires concomitant activation of diverse signaling pathways. Availability of stable, preassembled matrices at the plasma membrane would facilitate scaffolding of a large array of receptors, coreceptors, tyrosine kinases and other signaling and adapter proteins, as it is the case in signaling via the T cell antigen receptor. The concept of the signaling platform has gained usage to characterize the membrane structure where many different membrane-bound components need to be assembled in a coordinated manner to carry out signaling. The structural basis of the signaling platform lies in preferential assembly of certain classes of lipids into distinct physical and functional compartments within the plasma membrane. These membrane microdomains or rafts (Figure 1) serve as privileged sites where receptors and proximal signaling molecules optimally interact. In this review, we shall discuss first how signaling platforms are assembled and how receptors and their signaling machinery could be functionally linked in such structures. The second part of our review will deal with selected examples of raft-based signaling pathways in T lymphocytes and NK cells to illustrate the ways in which rafts may facilitate signaling.

Functional Characterization of the Intermediate Isoform of the Human Prolactin Receptor

Prolactin-dependent signaling occurs as the result of ligand-induced dimerization of the prolactin receptor (PRLr). While three PRLr isoforms have been characterized in the rat, studies have suggested the existence of several human isoforms in breast carcinoma species and normal tissues. Reverse transcription polymerase chain reaction was performed on mRNA isolated from the breast carcinoma cell line T47D, revealing two predominant receptor isoforms: the previously described long PRLr and a novel human intermediate PRLr. The nucleotide sequence of the intermediate isoform was found to be identical to the long isoform except for a 573-base pair deletion occurring at a consensus splice site, resulting in a frameshift and truncated intracytoplasmic domain. Scatchard analysis of the intermediate PRLr revealed an affinity for PRL comparable with the long PRLr. While Ba/F3 transfectants expressing the long PRLr proliferated in response to PRL, intermediate PRLr transfectants exhibited modest incorporation of [(3)H]thymidine. Significantly, however, both the long and intermediate PRLr were equivalent in their inhibition of apoptosis of the Ba/F3 transfectants after PRL treatment. The activation of proximal signaling molecules also differed between isoforms. Upon ligand binding, Jak2 and Fyn were activated in CHO-K1 cells transiently transfected with the long PRLr. In contrast, the intermediate PRLr transfectants showed equivalent levels of Jak2 activation but only minimal activation of Fyn. Last, Northern analysis revealed variable tissue expression of intermediate PRLr transcript that differed from that of the long PRLr. Taken together, differences in signaling and tissue expression suggest that the human intermediate PRLr differs from the long PRLr in physiological function.

A transgenic mouse model to analyze CD8(+) effector T cell differentiation in vivo.

Antigen-specific effector T cells are prerequisite to immune protection, but because of the lack of effector cell-specific markers, their generation and differentiation has been difficult to study. We report that effector cells are highly enriched in a T cell subset that can be specifically identified in transgenic (T-GFP) mice expressing green fluorescent protein (GFP) under control of the murine CD4 promoter and proximal enhancer. Consistent with previous studies of these transcriptional control elements, GFP was strongly and specifically expressed in nearly all resting and short-term activated CD4(+) and CD8(+) T cells. However, when T-GFP mice were challenged with vaccinia virus, allogeneic tumor cells, or staphylococcal enterotoxin A, the cytotoxic and IFN-gamma-producing T cells lost GFP expression. Upon T cell receptor (TCR) ligation by alphaCD3, sorted GFP(+) cells fluxed calcium and proliferated vigorously. In contrast, GFP(-) effector cells showed a diminished calcium flux and did not proliferate. Instead, they underwent apoptosis unless supplied with exogenous IL-2. By reverse transcription-PCR analysis, the GFP(-) cells up-regulated the pro-apoptotic molecule, Fas-L, and down-regulated gene expression of the proximal TCR signaling molecule, CD3zeta, and c-jun, a component of the AP-1 transcription factor. Thus, differential regulation of TCR signaling may explain the divergent responses of naïve and effector T cells to antigen stimulation.

The alpha 1A-adrenergic receptor subtype mediates biochemical, molecular, and morphologic features of cultured myocardial cell hypertrophy

alpha 1-Adrenergic agonists induce a hypertrophic phenotype in cultured neonatal rat ventricular myocytes. Quantifiable markers of this phenotype include stimulation of phosphoinositide hydrolysis, transcriptional induction of atrial natriuretic factor (ANF) gene expression, and an increase in myocardial cell size. The aim of the present work was to determine which alpha 1-adrenergic receptor subtype mediates the acquisition of these parameters of myocardial cell hypertrophy. Phosphoinositide hydrolysis is inhibited by low concentrations of 5-methylurapidil (log Ki = -8.7) and (+)-niguldipine (log Ki = -10.6). The alpha-adrenergic receptor-induced increase in transcriptional activation of an ANF luciferase reporter gene is inhibited over the same range of concentrations of 5-methylurapidil (log Ki = -8.2) and (+)-niguldipine (log Ki = -11.2) that inhibit phosphoinositide hydrolysis. In addition, the increase in cell size that accompanies alpha-adrenergic receptor stimulation of cultured ventricular myocytes is blocked by similar concentrations of 5-methylurapidil (log Ki = -8.0) and (+)-niguldipine (log Ki = -10.6). In contrast, treatment with the alpha 1B selective antagonist chlorethylclonidine at a concentration of 10 microM had no effect on the adrenergically mediated induction of ANF luciferase reporter gene expression or the adrenergically induced increase in myocardial cell size. These findings demonstrate that pharmacologically identifiable alpha 1A-adrenergic receptors mediate not only the early effects of alpha 1-adrenergic stimulation such as phosphoinositide hydrolysis, but that they activate the signaling pathways that control transcriptional induction of the ANF luciferase reporter gene and an increase in myocardial cell size. Studies using alpha 1-adrenergic receptor cDNAs to delineate and alter the direct interaction of this receptor subtype with proximal signaling molecules, e.g. GTP binding proteins, should provide a powerful means of assessing their role in the induction of the molecular and morphologic parameters of myocardial cell hypertrophy.

A study of patients attending without appointments in an urban general practice.

To ascertain which social and psychological characteristics are associated with patients attending surgeries without appointments. Prospective study of patients attending an urban centre group practice. Urban health centre group practice with five doctors and 12,000 patients in an area of high (greater than 20%) unemployment and social deprivation. All attenders at the open access surgery and one in four consecutive attenders by appointment, selected sequentially from the first three appointments, during 10 days in January 1989. Patients participating in the pilot study, reattending during the study period, or attending antenatal clinics were excluded. Patients' attitude to making appointments and reasons for attending, including perception of urgency, with respect to sociodemographic and psychosocial data obtained from a self completed questionnaire before the consultation. Doctors' diagnosis and perception of urgency obtained from a separate questionnaire. 86% (141/172) Of patients attending without appointments and 96% (139/145) with appointments responded to the questionnaire. The need for consultation was considered to be "very urgent" or "fairly urgent" in significantly more of the open access group than the appointments group (89%, 124/139 v 66%, 91/138; chi 2 = 27.04, df = 3; p less than 0.001), although the doctors did not share the same views. Significantly more patients had self limiting conditions of recent onset in the open access than in the appointments group (75%, 101/135 v 48%, 59/123: p less than 0.001). Overall, open access attendance was significantly linked with social support (39%, 48/124 v 26%, 32/123; p less than 0.05) and with marital separations or intentions to separate (10%, 9/87 v 0/92; 47%, 32/87 v 22%, 20/92 respectively; both p less than 0.001), but the doctors recorded significantly fewer psychological and social problems in these patients (p less than 0.05). Although almost half those in the appointments group considered that making appointments was inconvenient, more of those in the open access group agreed with this view (47%, 60/129 v 61%, 80/131). There was an important link between social support problems and a negative attitude to making appointments. In our previous experience encouraging patients to make appointments has been unsuccessful; practices serving areas with a high prevalence of social deprivation providing a mixed open access and appointments system may better serve patients' needs.