Insulin stimulates sequestration of beta-adrenergic receptors and enhanced association of beta-adrenergic receptors with Grb2 via tyrosine 350

G-protein-linked receptors, such as the beta2-adrenergic receptor, are substrates for growth factor receptors with intrinsic tyrosine kinase activity (Karoor, V., Baltensperger, K., Paul, H., Czech, M. P., and Malbon C. C. (1995) J. Biol. Chem. 270, 25305-25308). In the present work, the counter-regulatory action of insulin on catecholamine action is shown to stimulate enhanced sequestration of beta2-adrenergic receptors in either DDT1MF-2 smooth muscle cells or Chinese hamster ovary cells stably expressing beta2-adrenergic receptors. Both insulin and insulin-like growth factor-1 stimulate internalization of beta-adrenergic receptors, contributing to the counter-regulatory effects of these growth factors on catecholamine action. In combination with beta-adrenergic agonists, insulin stimulates internalization of 50-60% of the complement of beta-adrenergic receptors. Insulin administration in vitro and in vivo stimulates phosphorylation of Tyr-350 of the beta-adrenergic receptor, creating an Src homology 2 domain available for binding of the adaptor molecule Grb2. The association of Grb2 with beta-adrenergic receptors was established using antibodies to Grb2 as well as a Grb2-glutathione S-transferase fusion protein. Insulin treatment of cells provokes binding of Grb2 to beta2-adrenergic receptors. Insulin also stimulates association of phosphatidylinositol 3-kinase and dynamin, via the Src homology 3 domain of Grb2. Both these interactions as well as internalization of the beta-adrenergic receptor are shown to be enhanced by insulin, beta-agonist, or both. The Tyr-350 --> Phe mutant form of the beta2-adrenergic receptor, lacking the site for tyrosine phosphorylation, fails to bind Grb2 in response to insulin, fails to display internalization of beta2-adrenergic receptor in response to insulin, and is no longer subject to the counter-regulatory effects of insulin on cyclic AMP accumulation. These data are the first to demonstrate the ability of a growth factor insulin to counter-regulate G-protein-linked receptor, the beta-adrenergic receptor, via a new mechanism, i.e. internalization.     

Heterologous regulation of muscarinic and beta-adrenergic receptors in rat cardiomyocytes in culture

Previous work indicated that hyperstimulation of muscarinic receptors brings about profound changes not only in the density of the muscarinic receptors, but also of the beta-adrenoceptors in rat heart atria in vivo. We have now investigated whether a similar receptor cross-regulation occurs in cardiomyocytes in vitro. Cardiomyocytes from 3-4 day old rats were exposed to chemical agents on days 5-6 in culture. Densities of muscarinic and beta-adrenergic receptors were measured according to the binding of N-[3H]methylscopolamine and [ H]CGP 12177, respectively, to cell surface membranes and cell homogenates. Exposure of cells to the muscarinic agonist carbachol (1 mmol/l) brought about a profound decrease in the number of muscarinic receptors. The number of beta-adrenoceptors displayed biphasic changes, being augmented after 24 h (by 20-45% on the cell surface and by 29% in the homogenate) and diminished after 48 h and 72 h (after 48 h, decrease by 44-75% on the cell surface and by 36% in the homogenate). These effects of carbachol were not prevented by dimethylaminopropyl-bis-indolylmaleimide, the inhibitor of protein kinase C. Exposure of cells to the beta-adrenoceptor agonist isoprenaline (0.1 mmol/l) strongly diminished the number of beta-adrenoceptors on the cell surface and in the homogenate. The density of muscarinic receptors on the cell surface was diminished by 24-43% after 24 h exposure to isoprenaline and unchanged after 48 h, whereas the concentration of muscarinic receptors in the homogenate was unchanged after 24 h and increased by 20% after 48 h. The isoprenaline-induced decrease in the density of cell surface muscarinic receptors could not be simulated by forskolin and was not abolished by the protein kinase A inhibitors Rp-cAMPS and HA-1004. Dibutyryl cyclic AMP diminished the density of cell surface muscarinic receptors more than that of the beta-adrenergic receptors. Our data reveal a novel phenomenon of a biphasic change (an increase followed by a loss) in the density of beta-adrenoceptors during exposure of cardiocytes to carbachol. Activation of beta-adrenoceptors brings about less conspicuous changes in the density of muscarinic receptors. The observed phenomena of receptor cross-regulation cannot be explained by simple activations of protein kinases A and C.     

Agonist action at D2(long) dopamine receptors: ligand binding and functional assays

Clinical pathways are being introduced by hospitals to reduce costs and control unnecessary variation in care. We studied 766 inpatients to measure the impact of a perioperative clinical pathway for patients undergoing knee replacement surgery on hospital costs. One hundred twenty patients underwent knee replacement surgery before the development of a perioperative clinical pathway, and 63 patients underwent knee replacement surgery after pathway implementation. As control groups, we contemporaneously studied 332 patients undergoing radical prostatectomy (no clinical pathway in place for these patients) and 251 patients undergoing hip replacement surgery without a clinical pathway (no clinical pathway and same surgeons as patients having knee replacement surgery). Total hospitalization costs (not charges), excluding professional fees, were computed for all patients. Mean (+/-SD) hospital costs for knee replacement surgery decreased from $21,709 +/- $5985 to $17,618 +/- $3152 after implementation of the clinical pathway. The percent decrease in hospitalization costs was 1.56-fold greater (95% confidence interval 1.02-2.28) in the knee replacement patients than in the radical prostatectomy patients and 2.02-fold greater (95% confidence interval 1.13-5.22) than in the hip replacement patients. If patient outcomes (e.g., patient satisfaction) remain constant with clinical pathways, clinical pathways may be a useful tool for incremental improvements in the cost of perioperative care. Implications: Doctors and nurses can proactively organize and record the elements of hospital care results in a clinical pathway, also known as "care pathways" or "critical pathways." We found that implementing a clinical pathway for patients undergoing knee replacement surgery reduced the hospitalization costs of this surgery.     

Visualization of agonist-induced sequestration and down-regulation of a green fluorescent protein-tagged beta2-adrenergic receptor

To date, the visualization of beta2-adrenergic receptor (beta2AR) trafficking has been largely limited to immunocytochemical analyses of acute internalization events of epitope-tagged receptors in various transfection systems. The development of a beta2AR conjugated with green fluorescent protein (beta2AR-GFP) provides the opportunity for a more extensive optical analysis of beta2AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of beta2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events. Time-dependent colocalization of beta2AR-GFP with rhodamine-labeled transferrin and rhodamine-labeled dextran following agonist exposure demonstrates receptor distribution to early endosomes (sequestration) and lysosomes (down-regulation), respectively. The observed temporal distribution of beta2AR-GFP was consistent with measures of receptor sequestration and down-regulation generated by radioligand-receptor binding assays. Cells stimulated with different beta-agonists revealed time courses of beta2AR-GFP redistribution reflective of the intrinsic activity of each agonist.     

Direct binding and functional transfer of NK cell inhibitory receptors reveal novel patterns of HLA-C allotype recognition

Cytotoxicity of human NK cells is under negative control of killer cell Ig-like receptors (KIR) specific for HLA class I. To determine the specificity of five KIR containing two Ig domains (KIR2D), direct binding of soluble recombinant KIR2D to a panel of HLA class I transfectants was assayed. One soluble KIR2D, derived from an inhibitory receptor with a long cytoplasmic tail (KIR2DL1), bound to HLA-C allotypes containing asparagine 77 and lysine 80 in the heavy chain, as expected, since these allotypes inhibit lysis by NK cells expressing KIR2DL1. Surprisingly, another KIR2D (KIR2DL2), which inhibits NK lysis of cells expressing HLA-C molecules with serine 77 and asparagine 80, bound to HLA-C allotypes carrying either amino acid motif. Expression of the KIR2DL receptors in NK cells using recombinant vaccinia viruses confirmed these patterns of recognition, and identified KIR2DL3 as another KIR reacting with both groups of HLA-C allotypes. Mutagenesis of amino acid 44 in KIR2DL1 and KIR2DL2 suggested this residue controls the affinity of KIR for the 77/80 motif of HLA-C molecules. Two other soluble KIR2D, derived from noninhibitory receptors with short cytoplasmic tails (KIR2DS), did not bind to any of the HLA class I allotypes tested. One of these receptors (KIR2DS2) is closely related in sequence to KIR2DL2. Substitution of tyrosine 45 with the phenylalanine conserved in other KIR was sufficient to permit specific binding of KIR2DS2 to HLA-C. These results show that KIR2DL receptors are specific for HLA-C, but that recognition of HLA-C allotypes appears more permissive than indicated by previous functional experiments.     

Guanine nucleotide regulation and cation sensitivity of agonist binding to rat brain oxytocin receptors

The neuropeptide oxytocin (OT) is synthesized in the hypothalamus and can be released either as a hormone from the neurohypophysis or as a neurotransmitter in various brain regions. The present studies were undertaken to better characterize the pharmacological properties of brain oxytocin receptors (OTRs) using a radioligand selective for OTRs. Based on kinetic analysis, brain membranes obtained from 10-day-old rats display rapid and reversible binding to this ligand. In addition, saturation isotherm studies demonstrated that binding was saturable and of high affinity. Indicative of the selectivity of these receptors, compounds known to be ligands for OTRs in other tissues were able to displace the radioligand with high affinity. Consistent with the divalent cation requirement of OTRs in other tissues, OT binding was greatly reduced in rat brain membranes by the removal of magnesium from the incubation. To examine the possible GTP regulation of these receptors, binding was examined in the presence of a GTP analog. High affinity agonist, but not antagonist, binding was reduced by the GTP analog, indicating that these OTRs are likely to be associated with G proteins.     

Localization by indirect immunofluorescence of nitric oxide synthase in mouse and human spermatozoa

OBJECTIVE: To determine and compare the concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) in the peritoneal fluid of women with and without endometriosis. DESIGN: Prospective study. SETTING: Tertiary care center affiliated with university medical school. PATIENTS: Forty-two women with endometriosis and 34 women without endometriosis confirmed by laparoscopy. MAIN OUTCOME MEASURES: Epidermal growth factor and basic FGF concentrations were determined by highly sensitive enzyme immunoassays using monoclonal antibodies. RESULT: Approximately 50% of the PF samples had EGF concentrations of > 0.4 pg/mL (conversion factor to SI unit, 0.17) and 90% had basic FGF concentrations of > 1 pg/mL (conversion factor to SI unit, 0.058). Concentrations of EGF or basic FGF in PF did not differ significantly between the two groups. In normal women, there was a significant correlation between EGF and basic FGF during the luteal phase. In women with endometriosis, EGF levels were higher during the luteal phase, but there was no correlation of either EGF or basic FGF levels with the severity of endometriosis. CONCLUSIONS: The concentrations of EGF and basic FGF were highly variable in the PF of women with or without endometriosis and did not differ significantly. The concentrations were so low that neither EGF nor basic FGF in PF could bind to its receptor.     

A case of nonscarring inflammatory epidermolysis bullosa acquisita: characterization of IgG autoantibodies by immunofluorescence, immunoblotting and immunogold electron microscopy

We report a case of nonscarring inflammatory epidermolysis bullosa acquisita in a 59-year-old Japanese woman. She developed blisters and erosions on her lip, trunk and extremities. Sodium aurothiomalate was effective for the skin lesions. The patient had been free from bullous skin lesions for the last 13 years and had shown no scarring. Indirect immunofluorescence (IF) study on 1 M NaCl-split skin revealed IgG autoantibodies against the dermal side of the split skin. Immunoblotting using normal human dermal extracts disclosed IgG autoantibodies reactive with the 290 and 145 kD antigens. Circulating IgG autoantibodies were deposited on the lamina densa by immunoelectron microscopy. IF mapping using several antibodies for the components of the basement membrane zone revealed blister formation at the lamina densa. These results suggest that the cleavage at the lamina lucida does not necessarily exclude the diagnosis of EBA and that the definite diagnosis of EBA should be confirmed by immunoblotting or immunoelectron microscopic study.     

Agonist and antagonist effects of apomorphine enantiomers on 5-HT3 receptors

Human platelets express several sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) isoenzymes: SERCA2b of 100 kDa apparent molecular mass and two distinct enzymes of 97 kDa, one of them identified as being the SERCA3a isoform. The molecular identity of the third enzyme specifically recognized by the PL/IM430 monoclonal antibody has remained elusive. First, the study of the 3'-end part of platelet SERCA3 mRNA, by means of RT-PCR amplification using sets of primers covering the N-3 to N (ultimate) exons of the human SERCA3 sequence, revealed the presence of two distinct mRNA sequences, SERCA3a and a longer variant. Second, this additional sequence was identified as SERCA3b and found to refer to the insertion of a new exon of 73 bp, located at bp 349 from the beginning of the intronic sequence, linking the penultimate (N-1) exon to the last exon (N) of the human SERCA3 gene. Third, a relationship between the expression of this SERCA3b mRNA and the PL/ IM430 recognizable SERCA protein was observed. SERCA3b mRNA was found to be absent in epithelial HeLa cells not recognized by the PL/IM430 antibody and the expression of this SERCA3b RNA species correlated with that of the SERCA protein recognized by PL/IM430 which was down-modulated in the platelet precursor megakaryocytic CHRF 288-11 cell line as well as upon in vitro lymphocyte activation. Taken together, these results strongly support the notion of the presence of the SERCA3b protein in human cells by showing SERCA3b mRNA in platelets and the fact that the protein corresponding to this mRNA species is very likely the 97 kDa protein recognized by the PL/IM430 antibody.     

Dopamine agonist-induced hypothermia and disruption of prepulse inhibition: evidence for a role of D3 receptors?

The dopamine D3/D2 receptor agonists 7-OH-DPAT, quinpirole, quinelorane, and PD128907, the mixed dopamine agonist apomorphine, the D2 agonist bromocriptine, and the D1/D5 agonist SKF38393 were examined in models of hypothermia and prepulse inhibition (PPI) in Wistar rats. As dopamine agonist-induced hypothermia has been proposed as a model of D3 receptor function, and dopamine agonists are known to disrupt PPI, drug potencies to induce hypothermia were established and compared with doses necessary to disrupt PPI. 7-OH-DPAT, quinpirole, quinelorane, PD128907, and apomorphine, reduced body temperature and disrupted PPI with a similar rank order of potency (quinelorane > quinpirole = 7-OH-DPAT > PD128907 = apomorphine). Bromocriptine and SKF38393 were ineffective in both models. In a separate study, the dopamine reuptake inhibitors cocaine and GBR 12909 had no effect on PPI. In a final set of studies, the D2/D3 antagonist raclopride blocked both 7-OH-DPAT-induced hypothermia and 7-OH-DPAT-induced PPI disruption. The 5-HT1A antagonist WAY 100,135, and the peripheral D2-like antagonist domperidone had no effect. These findings suggest that the hypothermia and PPI disruptions seen with some of these dopamine agonists may be mediated by central D3 receptors; however, only studies using more selective dopamine receptor ligands can definitively rule out effects at the D2 or D4 receptors.     

CD4:p56lck association studied in vivo using antibody-induced capping and double indirect immunofluorescence microscopy

Accumulating data suggest that the T-cell surface antigen CD4 transduces an independent signal during antigen-mediated T-cell activation. In vitro studies which showed that the cytoplasmic protein tyrosine kinase p56lck is present in anti-CD4 immunoprecipitates led to the model that p56lck is associated with the cytoplasmic domain of CD4. In this report we have extended these studies and examined potential CD4:p56lck associations in vivo. We show here by double immunofluorescence microscopy a specific co-distribution of p56lck with antibody-induced CD4 caps in intact cells. Murine T-cell hybridoma lines expressing mutant forms of CD4 were used to demonstrate that the 31 carboxyterminal aminoacids of its cytoplasmic domain, in particular cysteine-420 and cysteine-422, are crucial for the formation of CD4:p56lck complexes in vivo. The potential of the method applied is discussed with regard to studies of other transmembrane signalling systems involving src-like kinases.     

Cryptosporidium parvum in patients with chronic diarrhea and AIDS: diagnosis by means of indirect immunofluorescence with monoclonal antibodies

The diagnosis of cryptosporidiosis is difficult when oocyst elimination is poor as occurs in AIDS patients. Aiming to improve the diagnosis, 144 fecal samples coming from AIDS patients with diarrhea, were studied using indirect immunofluorescence with anti-Cryptosporidium monoclonal antibodies. The results were compared with Ziehl Neelsen and safranine stainings. Twenty three samples (15.9%) were positive for Cryptosporidium with at least one of the three methods. Sensitivities were 78.3% for immunofluorescence, 86.9% for Ziehl Neelsen and 91.3 for safranine stainings. The specificity of the three methods was 100%. It is concluded that immunofluorescence does not improve the diagnostic accuracy of cryptosporidiosis and its high cost precludes its use in routine laboratories.     

Characterization of beta-adrenergic receptors of freshly isolated astrocytes and neurons from rat brain

The binding and characteristics of rat brain beta-adrenergic receptors (beta-AR) isolated from astrocytes and neurons were investigated. Equilibrium binding experiments demonstrated that beta-AR were more concentrated on astrocytes than on neurons isolated from forebrain, cerebral cortex and cerebellum. Inhibition experiments revealed that beta 1-AR and beta 2-AR were present in the two cell types. Isoproterenol revealed two interchangeable states of high and low affinity binding to both beta 1- and beta 2-AR in neurons. The high affinity binding sites were sensitive to guanylylimidodiphosphate (GppNHp). Similar results were found with other beta-AR agonists but not with salbutamol and salmeterol which recognized both affinity states of the neuronal beta 2-AR but only the low affinity state of beta 1-AR. In astrocytes only the low affinity state of beta-AR was observed.     

Progesterone receptors: expression and regulation in the mammalian ovary

The authors have briefly discussed the molecular structure, regulation, and function of progesterone receptors in the mammalian ovary. Particularly important is the contrast in the regulatory mechanisms of PR induction in the ovary (gonadotropins/membrane receptor mediated) and other well-known progesterone target tissues, such as the uterus and mammary gland (estrogen/nuclear receptor mediated). Future research will focus on how the PR gene responds to these hormonal regulatory signals in this cell-specific manner. Equally important in this discussion has been the mounting evidence indicating that PRs are an essential component of the ovulatory process. The observation that PR-/- knockout mice are incapable of undergoing ovulation, even in response to gonadotropin challenge, further supports the previous physiological evidence indicating that PRs in preovulatory follicles are induced before, and are necessary for, ovulation. Further studies are required to determine the identity of PR-regulated target genes during the periovulatory period. Although our knowledge of PR structure, regulation, and function has increased dramatically during the past decade, many exciting questions remain related to the regulation and function(s) of PRs in the ovary and other tissues.     

Regulation of beta-adrenergic receptors by guanyl-5'-yl imidodiphosphate and other purine nucleotides

Guanyl-5'-yl imidodiphosphate (Gpp(NH)p), GTP, and other purine nucleotides selectively decrease the binding affinity of the beta-adrenergic receptors of frog erythrocyte membranes for beta-adrenergic agonists but not antagonists. Shifts in binding affinity were assessed by determining the ability of unlabeled ligands to compete with (-)-[3H]dihydroalprenolol for the membrane-bound receptors. The magnitude of the"right" shift in the binding displacement curve for any of 13 ligands tested was directly related to the intrinsic activity (maximal stimulatory capacity) of that agent for stimulation of the frog erythrocyte membrane adenylate cyclase. Thus, Gpp(NH)p-induced shifts in binding affinity were greatest for full agonists such as isoproterenol, intermediate for partial agonists such as soterenol, and no shifts were observed for antagonists such as propranolol. Shifts in binding affinity were observed only in preparations where agonist binding to the receptors leads to "coupling" of the receptors with adenylate cyclase. In solubilized preparations where the beta-adrenergic receptors and adenylate cyclase are functionally "uncoupled", Gpp(NH)p did not cause right shifts in agonist receptor binding displacement curves. In particulate preparations the Km of Gpp(NH)p for stimulation of adenylate cyclase was identical with that for its effect on beta-adrenergic agonist binding affinity, 1 to 2 muM. Moreover, the ability of several other nucleotides to cause shifts in receptor binding affinity directly paralleled their previously determined affinities for the nucleotide regulatory sites on adenylate cyclase. Gpp(NH)p also shifted agonist dose-response curves for stimulation of adenylate cyclase, but to the left. As with the effects on the receptor binding curves, the effects of Gpp(NH)p on the "apparent affinities" of agonists for enzyme stimulation were directly related to their intrinsic activities. Gpp(NH)p also markedly increased the intrinsic activity of partial agonists. These results appear to indicate that conformational alterations in adenylate cyclase caused by occupation of nucleotide regulatory sites by Gpp(NH)p are capable of inducing alterations in the beta-adrenergic receptors. These receptor alterations are induced only when the receptors are "coupled" to the enzyme by virtue of agonist binding. The nucleotide-altered conformation of the beta-adrenergic receptors is characterized by decreased binding affinity for agonist but increased functional efficacy in stimulating the enzyme.     

Clinical comparison of borreliacidal-antibody test with indirect immunofluorescence and enzyme-linked immunosorbent assays for diagnosis of Lyme disease

OBJECTIVE: To compare the clinical results with the borreliacidal-antibody test (BAT) and two standard screening serologic tests for Lyme disease (LD)-the indirect immunofluorescence assay (IFA) and the enzyme-linked immunosorbent assay (ELISA). DESIGN: The medical records of patients from an endemic LD area, who had been serologically tested during the summer of 1992, were retrospectively categorized by clinical diagnoses without results of serologic tests. Serologic testing, which included control serum samples from patients from a nonendemic LD area, was performed in a blinded fashion, and the results were compared with the clinical categories. MATERIAL AND METHODS: Medical records of 307 patients who had been serologically tested for LD were reviewed. We found untreated, active LD in 43 patients (early-localized LD, 21; early-disseminated LD, 14; and late-disseminated LD, 8) and treated LD in 33. Non-LD cases were categorized into acute or chronic conditions of unknown or known cause. RESULTS: Overall, the BAT had a sensitivity of 11% in active LD and did not correlate with results of other conventional surface antibody assays. The IFA and ELISA were more sensitive (67 to 93%), but false-positive results frequently were noted (20 to 40%) in acute and chronic non-LD inflammatory conditions. The specificity of the BAT, IFA, and ELISA in the control group was 96%, 93%, and 97%, respectively. CONCLUSION: Until the sensitivity, as measured by prospective clinical studies, is improved without loss of specificity, the BAT should not be used clinically for the diagnosis of LD. Suspected cases of LD with atypical clinical manifestations should have positive ELISA and IFA results confirmed with a standardized immunoblot assay.     

Scavenger receptors: diverse activities and promiscuous binding of polyanionic ligands

Scavenger receptors are a diverse family of proteins that share a common property--the binding of modified lipoprotein--but they have recently been shown to recognise a diverse range of ligands. Understanding the molecular interaction of receptor-ligand binding should provide insight into how scavenger receptors contribute to important biological processes.