Lymphoma Cell Membranes Research Articles

Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.

Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.

Combination of cytokine-induced killer and dendritic cells pulsed with antigenic α-1,3-galactosyl epitope–enhanced lymphoma cell membrane for effective B-cell lymphoma immunotherapy

Background aimsRefractory B-cell lymphomas are difficult to successfully treat with current chemotherapeutic regimens; however, immunotherapy may be an effective form of treatment for these patients. MethodsFourteen refractory lymphoma patients (age, 29–74 y) were enrolled in the trial. α-1,3-galactosyl (α-Gal) epitopes were synthesized on lymphoma cell membranes with the use of bovine recombinant α-1,3-galactosyltransferase (α-GT) and neuraminidase to enhance tumor immunogenicity. Subsequent incubation of processed cell membranes with autologous dendritic cells (DCs) in the presence of human serum containing abundant natural anti–α-Gal immunoglobulin G led to the effective phagocytosis of tumor membranes by DCs. The pulsed DCs and autologous cytokine-induced killer cells were then co-cultured to promote maximum cytotoxicity to lymphoma cells and were infused back into the donor lymphoma patients. Therapeutic responses were assessed by clinical observation, laboratory tests and a computed tomography scan at 6 months after treatment. ResultsComplete and partial remission occurred in four and three patients, respectively. The disease status remained unchanged in five patients, and disease progression was observed in two patients. No serious side effects or autoimmune diseases were observed in any participants. Serum lactate dehydrogenase and β2-macroglobulin decreased in 11 and 14 patients, respectively. All patients showed robust systemic cytotoxicity in response to tumor lysate as measured by interferon-γ expression in peripheral blood mononuclear cells after treatment (P < 0.001). The number of peripheral immune effector cells (CD3+/CD4+, CD8+/CD28+ and CD16+/CD56+ cells) increased significantly (P < 0.05) 3 months after treatment. ConclusionsLymphoma cell–specific α-Gal immunotherapy is safe, effective and has great potential for the treatment of refractory B-cell lymphoma.

Targeting IL11 Receptor in Leukemia and Lymphoma: A Functional Ligand-Directed Study and Hematopathology Analysis of Patient-Derived Specimens.

The IL11 receptor (IL11R) is an established molecular target in primary tumors of bone, such as osteosarcoma, and in secondary bone metastases from solid tumors, such as prostate cancer. However, its potential role in management of hematopoietic malignancies has not yet been determined. Here, we evaluated the IL11R as a candidate therapeutic target in human leukemia and lymphoma. First, we show that the IL11R protein is expressed in a variety of human leukemia- and lymphoma-derived cell lines and in a large panel of bone marrow samples from leukemia and lymphoma patients, whereas expression is absent from nonmalignant control bone marrow. Moreover, a targeted peptidomimetic prototype (termed BMTP-11), specifically bound to leukemia and lymphoma cell membranes, induced ligand-receptor internalization mediated by the IL11R, and resulted in a specific dose-dependent cell death induction in these cells. Finally, a pilot drug lead-optimization program yielded a new myristoylated BMTP-11 analogue with an apparent improved antileukemia cell profile. These results indicate (i) that the IL11R is a suitable cell surface target for ligand-directed applications in human leukemia and lymphoma and (ii) that BMTP-11 and its derivatives have translational potential against this group of malignant diseases.

Rituximab activates Syk and AKT in CD20-positive B cell lymphoma cells dependent on cell membrane cholesterol levels

The introduction of rituximab, an anti-CD20 monoclonal antibody, has dramatically improved the treatment outcomes of patients with B cell lymphoma. Nevertheless, the clinical response to rituximab varies, and a subpopulation of patients does not respond well to this antibody. Although several molecular events have been shown to be involved in the mechanism of action of rituximab, recent studies have demonstrated that intracellular signaling pathways and the direct effects of rituximab on cell membrane components are responsible for the antilymphoma action of this drug. In the present study, we demonstrated that rituximab activated Syk and Akt, molecules with antiapoptotic functions, in several CD20-positive lymphoma cell lines. Notably, rituximab activated Syk and Akt in all the tested primary lymphoma samples from six patients. Our results show that the cholesterol levels in lymphoma cell membranes have a crucial role in the regulation of Syk and Akt. The depletion of cholesterol from the cell membrane completely blocked rituximab-induced Syk and Akt activation. Simvastatin, an inhibitor of cholesterol synthesis, also abrogated rituximab-mediated Syk and Akt activation. Finally, we report that rituximab inhibited the apoptosis induced by chemotherapeutic drugs, which was observed solely in Akt-activated cells. This work demonstrates for the first time that rituximab paradoxically works to suppress apoptosis under certain conditions in a manner that is dependent on the cell membrane cholesterol level. Our observations provide novel insights and suggest that the cell membrane cholesterol level represents a new biomarker for predicting patient response to rituximab. Furthermore, the modulation of lipid rafts could provide a new strategy for enhancing the antilymphoma action of rituximab.

Immunotherapy with autologous tumour antigen‐coated microbeads (large multivalent immunogen), IL‐2 and GM‐CSF in dogs with spontaneous B‐cell lymphoma

Cytotoxic T-lymphocyte responses to subcellular antigens are enhanced when antigens are presented on cell-sized silica microbeads called large multivalent immunogens (LMIs). LMIs prepared with tumour cell membrane fragments have induced partial remissions in humans with melanoma and renal cell carcinoma. The purpose of this phase I study was to evaluate the safety of LMIs, prepared with autologous lymphoma cell membranes, along with subcutaneous interleukin 2 (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF) in dogs with untreated B-cell lymphoma. After lymph node excision and induction chemotherapy, five dogs were vaccinated with three weekly doses of LMI alone; five with LMI and subcutaneous IL-2 and five with LMI, IL-2 and GM-CSF. No significant toxicity was noted, treatment did not adversely affect disease-free interval and half of the dogs showed measurable delayed-type hypersensitivity reactions to intradermal challenge with LMI, suggesting specific cell-mediated immunity.

Human γδ T lymphocytes strip and kill tumor cells simultaneously

When human γδ lymphocytes bind to tumor cells for killing, they also strip their membrane for unknown reasons. Here we investigated this topic using the model of human γδ lymphocytes co-incubated with anaplastic large cell lymphomas, a group of tumors with cytolytic T or null lineage. By using flow cytometry and live cell imaging, we show that as soon as both cells were in contact, the TCR-mediated activation of γδ lymphocytes simultaneously triggered their secretion of lytic granules and stripping of lymphoma cell membranes, and both activities continued even after their cell death. However reciprocally in such conjugates, resistant lymphoma failed to strip γδ cells and to kill them by untargeted secretion of their own lytic granules. This indicated that secretion of lytic granules and target membrane stripping are associated in lytic cell conjugates, and that γδ T lymphocytes strip and kill their targets simultaneously.

Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides

Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl(2). N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K(i) of 4.2 nm. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5'-[gamma-thio]triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5'-[gamma-thio]triphosphate and uridine 5'-[gamma-thio]triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC(50), 690 nm). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with K(i) values in the 200-400 nm range. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl(2) against MgCl(2) reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.

MANT-substituted guanine nucleotides: A novel class of potent adenylyl cyclase inhibitors

Mammals express nine membranous adenylyl cyclase (AC) isoforms (AC1-AC9), but the precise functions of AC isoforms are still incompletely understood. This situation is at least partially due to the paucity of potent and isoenzyme-specific AC inhibitors. The original aim of our research was to develop a fluorescence assay for the stimulatory G-protein of AC, G s. 2′(3′)- O-( N-methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and were previously used for the fluorescence analysis of purified G i/G o-proteins. We studied the effects of MANT-guanosine 5′-[γ-thio]triphosphate (MANT-GTPγS) and MANT-guanosine 5′-[β,γ-imido]triphosphate (MANT-GppNHp) on Gα s- and Gα i-mediated signaling. MANT-GTPγS and MANT-GppNHp had lower affinities for Gα s and Gα i than GTPγS and GppNHp. In contrast to guanosine 5′-[β-thio]diphosphate, MANT-GTPγS noncompetitively inhibited GTPγS-stimulated AC in Gα s-expressing Sf9 insect cell membranes. AC inhibition by MANT-GTPγS and MANT-GppNHp was not due to Gα s inhibition since it was also observed in Gα s-deficient S49 cyc − lymphoma cell membranes. Mn 2+ blocked Gα i-mediated AC inhibition by GTPγS and GppNHp in S49 cyc − membranes but not AC inhibition by MANT-GTPγS and MANT-GppNHp. MANT-GTPγS and MANT-GppNHp competitively inhibited forskolin/Mn 2+-stimulated AC in S49 cyc − membranes with K i values of 53 nM and 160 nM, respectively. Taken together, MANT-substituted guanine nucleotides constitute a novel class of potent competitive AC inhibitors. The availability of potent fluorescent AC inhibitors will help us study the kinetics of AC/nucleotide interactions as well as function, trafficking and localization of AC isoenzymes in intact cells. In future studies, we will examine the specificity of MANT-nucleotides for AC isoenzymes.

2′(3′)-O-(N-Methylanthraniloyl)-substituted GTP Analogs: A Novel Class of Potent Competitive Adenylyl Cyclase Inhibitors

2'(3')-O-(N-Methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and widely used for the kinetic analysis of enzymes and signaling proteins. We studied the effects of MANT-guanosine 5'-[gamma-thio]triphosphate (MANT-GTP gamma S) and MANT-guanosine 5'-[beta,gamma-imido]triphosphate (MANT-GppNHp) on G alpha(s)- and G alpha(i)-protein-mediated signaling. MANT-GTP gamma S/MANT-GppNHp had lower affinities for G alpha(s) and G alpha(i) than GTP gamma S/GppNHp as assessed by inhibition of GTP hydrolysis of receptor-G alpha fusion proteins. MANT-GTP gamma S was much less effective than GTP gamma S at disrupting the ternary complex between the formyl peptide receptor and G alpha(i2). MANT-GTP gamma S/MANT-GppNHp non-competitively inhibited GTP gamma S/GppNHp-, AlF(4)(-)-, beta(2)-adrenoceptor plus GTP-, cholera toxin plus GTP-, and forskolin-stimulated adenylyl cyclase (AC) in G alpha(s)-expressing Sf9 insect cell membranes and S49 wild-type lymphoma cell membranes. AC inhibition by MANT-GTP gamma S/MANT-GppNHp was not due to G alpha(s) inhibition because it was also observed in G alpha(s)-deficient S49 cyc(-) lymphoma cell membranes. Mn(2+) blocked AC inhibition by GTP gamma S/GppNHp in S49 cyc(-) membranes but enhanced the potency of MANT-GTP gamma S/MANT-GppNHp at inhibiting AC by approximately 4-8-fold. MANT-GTP gamma S and MANT-GppNHp competitively inhibited forskolin/Mn(2+)-stimulated AC in S49 cyc(-) membranes with K(i) values of 53 and 160 nm, respectively. The K(i) value for MANT-GppNHp at insect cell AC was 155 nm. Collectively, MANT-GTP gamma S/MANT-GppNHp bind to G alpha(s)- and G alpha(i)-proteins with low affinity and are ineffective at activating G alpha. Instead, MANT-GTP gamma S/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors.

Susceptibility of S49 lymphoma cell membranes to hydrolysis by secretory phospholipase A 2 during early phase of apoptosis

During cell death, plasma membranes of cells become vulnerable to attack by extracellular secretory phospholipase A 2. The purpose of this study was to identify the timing of this phenomenon relative to other events that occur during the process of cell death. Death was induced in S49 murine lymphoma cells by treatment with dexamethasone, dibutyryl cAMP, ionomycin, thapsigargin, or heat shock (1 h at 43°C). The appearance of membrane susceptibility to secretory phospholipase A 2 was compared to the following apoptotic events: loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, early DNA damage assessed by the comet assay, and changes in cell size and internal complexity assessed by flow cytometry. Each inducer of death was distinct in the time course of events produced. Although dead cells were susceptible to the action of phospholipase A 2, live cells (impermeable to propidium iodide) also became vulnerable to the enzyme during characteristic time courses after exposure to each inducer. In fact, susceptibility to sPLA 2 was observed in each case prior to or concurrent with the earliest of the markers of apoptosis. These results demonstrate that the onset of susceptibility to sPLA 2 is an early event in apoptosis suggesting that changes in membrane structure may be relevant to initial aspects of the apoptotic process.

Bovine CD18 identified as a species specific receptor for Pasteurella haemolytica leukotoxin

Ligand blotting of Pasteurella haemolytica leukotoxin (LKT) susceptible BL3 bovine lymphoma cell membranes with LKT detected two putative receptors with M r of 95 and 100 kDa, whereas no LKT binding to membrane proteins was detected for LKT non-susceptible human leukemic cells. Anti-bovine CD18 and CD11a/CD18 mAb recognized 95 and 100 kDa bands from BL3 cell membranes. CD18 isolated from BL3 cell membranes bound LKT. Pre-incubation of BL3 cells with anti-bovine CD18 or CD11a/CD18 mAb caused partial inhibition of LKT-induced leukolysis. Therefore, we propose that bovine CD18 acts as a species-specific leukocyte receptor for P. haemolytica LKT.

Altered G protein activity in a desensitization-resistant mutant of the Y1 adrenocortical tumor cell line.

Mutant isolates [designated desensitization resistant (DR)] from the Y1 mouse adrenocortical tumor cell line resist agonist-induced desensitization of adenylyl cyclase by preventing the uncoupling of receptors from their guanyl nucleotide-binding regulatory G proteins. In this study, we tested the hypothesis that an underlying G protein defect is associated with the DR phenotype. We found that the G protein reagent guanyl-5'-yl imidodiphosphate [Gpp(NH)p] shifted beta2-adrenergic receptors from a high affinity state to a low affinity state 4-fold more effectively in mutant DR cells than in parent Y1 cells. In the DR mutant, Gpp(NH)p was able to shift receptors to a low affinity state in the absence of NaCl, whereas the effect of Gpp(NH)p in parent Y1 cells was dependent upon the presence of NaCl. Moreover, these differences in sensitivity to Gpp(NH)p and NaCl were transferred to Gs alpha-deficient S49(CYC-) lymphoma cell membranes in G protein reconstitution assays. These observations suggested that the DR mutation was associated with altered activity of the stimulatory G protein, Gs. Cloning and sequence analysis demonstrated that Gs alpha transcripts in the DR mutant were normal, suggesting that another factor involved in guanyl nucleotide exchange is responsible for the altered G protein activity in DR mutant cells.

Enhancement of adenylate cyclase activity in S49 lymphoma cell membranes by the toxin thionin from Pyrularia pubera

W. Huang, L. P. Vernon and J. D. Bell. Enhancement of adenylate cyclase activity in S49 lymphoma cell membranes by the toxin thionin from Pyrularia pubera. Toxicon 32, 789–797, 1994.—We investigated the mode of action of Pyrularia thionin on adenylate cyclase in S49 lymphoma cell membranes. Thionin increased the forskolin-induced cAMP production in both wild-type and cyc - variant cell membranes. Thionin increased the apparent efficacy, but not potency, of the stimulation of adenylate cyclase by forskolin. Our results also indicated that the stimulatory G protein (G s) was not involved in the action of thionin on adenylate cyclase; however, thionin appeared to reduce the effect of G i on adenylate cyclase. We examined the effect of thionin on the temperature dependence of adenylate cyclase activity. The effect of thionin was not quantitatively the same at all temperatures. The effect was greatest in the range of about 30°C to 50°C. In addition, cardiotoxin, a structurally distinct peptide that alters the properties of biological membranes similarly to thionin, also enhanced the activity of adenylate cyclase. It seems reasonable to suspect that thionin influences the activity of adenylate cyclase indirectly by acting on the membrane.

Cardiac adenylyl cyclase, beta-adrenergic receptors, and G proteins in salt-sensitive hypertension.

The present study investigated whether high salt intake (8%) in Dahl salt-sensitive and salt-resistant rats with and without hypertension produces a heterologous desensitization of cardiac adenylyl cyclase as observed in various types of hypertension and human heart failure. In membranes from Dahl salt-sensitive rats on a high-salt diet (8%) basal, isoproterenol-, 5'-guanylylimidodiphosphate-, and forskolin-stimulated adenylyl cyclase was reduced compared with the low-salt (0.4%) group and Dahl salt-resistant rats on either 0.4% or 8% sodium chloride. The activity of the catalyst was depressed, and the expression of the immunodetectable inhibitory G proteins Gi alpha was increased in Dahl salt-sensitive rats on 8% sodium chloride, whereas the density of beta-adrenergic receptors and the activity of the stimulatory G protein Gs alpha reconstituted into Gs alpha-deficient S49 cyc- mouse lymphoma cell membranes were unchanged in any condition studied. We conclude that high salt intake in salt-sensitive hypertensive Dahl rats produces hypertension, cardiac hypertrophy, and heterologous desensitization of cardiac adenylyl cyclase. The latter alteration is due to an increase of Gi alpha proteins and a depressed catalyst activity of adenylyl cyclase. The results demonstrate that heterologous adenylyl cyclase desensitization can precede the development of contractile dysfunction in later stages and can occur independently of changes in beta-adrenergic receptors.

Inhibition of subunit dissociation and release of the stimulatory G-protein, Gs, by βγ-subunits and somatostatin in S49 lymphoma cell membranes

We examined the interaction between the stimulatory guanine-nucleotide-binding protein, Gs, and the inhibitory guanine-nucleotide-binding protein, Gi, in cell membranes of S49 lymphoma cells. In these cells, beta-adrenergic receptors stimulate the activity of adenylate cyclase via Gs, whereas inhibition via somatostatin receptors is transduced by an inhibitory G-protein, Gi. Using an antibody that selectively recognizes alpha s, the monomeric, but not the heterotrimeric, alpha-subunit of Gs, we quantified the extent of dissociation of Gs in a competitive e.l.i.s.a. Incubation of S49-cell plasma membranes with 0.1 microM-isoprenaline, 100 microM free Mg2+ and 100 microM-GTP produced substantial subunit dissociation of Gs, which was reversible by addition of purified beta gamma-subunit dimer or somatostatin. Somatostatin produced an immediate (without a lag) time- and concentration-dependent decrease in the concentration of dissociated Gs (kinhib. for somatostatin = 51 +/- 12 nM) and in the activity of adenylate cyclase (kinhib. = 121 +/- 20 nM). By contrast, after addition of a 10-fold molar excess of beta gamma-dimer relative to alpha s, there was a 2-3 min lag, after which the beta gamma-dimer re-associated Gs. Isoprenaline-induced dissociation of Gs was accompanied by a release of alpha s from the incubated membranes to a post-100,000 g supernatant, and somatostatin could reverse this release. Immunoblot analysis with both a C-terminal anti-peptide antibody and an antibody directed against a sequence near the N-terminal also showed release of alpha s by the beta-agonist and reversal by somatostatin. Membrane release of Gs by isoprenaline that could be blocked by somatostatin was also confirmed in reconstitution studies of supernatant fraction into cyc- S49-cell membranes. We conclude that in native cell membranes somatostatin-induced activation of Gi dissociates Gi and interferes with the Gs activation cycle by providing beta gamma-dimer, which acts to prevent or reverse formation of monomeric alpha s. Because alpha s can be released from the cell membrane, regulation of the local concentration of GTP-liganded dissociated alpha s is likely to be an important factor in modulating the activity of adenylate cyclase.

Inhibition of adenylate cyclase by p-bromophenacyl bromide

p-Bromophenacyl bromide (BPB) is an alkylating agent which has been used in biochemical studies as an inhibitor of phospholipase A 2 activity. We report here that BPB irreversibly inhibited adenylate cyclase activity stimulated by hormones, forskolin, GppNHp, NaF, and cholera toxin. The action of BPB in S49 lymphoma cell membranes (wild type and cyc −) indicates that it can inhibit adenylate cyclase function in the absence of G s. In the presence of G s, however, inhibition of adenylate cyclase by BPB was enhanced, suggesting that BPB may covalently modify the catalytic protein on a site involved in activated catalytic functioning or critical to its interaction with G s and/or additionally on the α s protein.

A Cytolysin, Θ-Toxin, Preferentially Binds to Membrane Cholesterol Surrounded by Phospholipids with 18-Carbon Hydrocarbon Chains in Cholesterol-Rich Region1

We have previously suggested the existence of two distinctive states of cholesterol in erythrocyte and lymphoma cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens [Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Ando, S., & Nagai, Y. (1988) Eur. J. Biochem. 176, 95-101; Ohno-Iwashita, Y., Iwamoto, M., Ando, S., Mitsui, K., & Iwashita, S. (1990) Biochim. Biophys. Acta 1023, 441-448]. To understand factor(s) which determine membrane cholesterol heterogeneity, we analyzed toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (phosphatidylcholine/phosphatidylglycerol = 82:18, mol/mol). Liposomes containing phospholipids with 18-carbon hydrocarbon chains at both positions 1 and 2 of the glycerol have both high- and low-affinity toxin-binding sites with Kd values similar to those of intact erythrocytes, whereas liposomes with hydrocarbon chains containing 16 or fewer carbons at either position 1 or 2 have only low-affinity toxin-binding sites. The cholesterol/phospholipid ratio, in addition to the length of phospholipid hydrocarbon chain, also determines the number of toxin-binding sites, indicating that at least these two factors determine the topology of membrane cholesterol by creating distinctively different affinity sites for the toxin. Since theta-toxin binding detects specific populations of membrane cholesterol that are not detectable by the measurements of susceptibility to cholesterol oxidase and cholesterol desorption from membranes, the toxin could provide a unique probe for studying the organization of cholesterol in membranes.

Pharmacological characterization of the novel helodermin/VIP receptor present in human SUP-T 1 lymphoma cell membranes

[Acetyl-His 1]VIP stimulated adenylate cyclase with higher potency than VIP in membranes from human SUP-T 1 lymphoblasts and was used as an efficient radioiodinated ligand with low non-specific binding to evaluate the relationship between receptor occupancy and adenylate cyclase activation and the possible interference of peptide T (an epitope derived from HIV envelope protein gp120). Various peptides inhibited [ 125I-acetyl-His 1]VIP binding and activated the enzyme, their order of potency being: helodermin > [acetyl-His 1]VIP > VIP = PHI = [Phe 1]VIP > [ d-Phe 2]VIP = [ d-Ala 4]VIP = [ d-Phe 4]PHI ≥ [ d-Phe 4]VIP > [ d-His 1]VIP giving further support for the existence of a novel subtype of helodermin/VIP receptors. [ d-Ala 1]peptide T and VIP-(10–28) did not recognize the binding site and did not inhibit, even at high concentration, VIP — or VIP analogue — stimulated adenylate cyclase activities.

Cell‐free heterologous desensitization of adenylyl cyclase in S49 lymphoma cell membranes mediated by cAMP‐dependent protein kinase

We have examined the cell-free heterologous desensitization of adenylyl cyclase in plasma membrane preparations from S49 wild-type (WT) and kin- cells (which lack cAMP-dependent protein kinase) incubated with purified catalytic subunit of cAMP-dependent protein kinase (cA.PKc). cA.PKc caused a rapid (t1/2 = 40 s) decrease in the hormone responsiveness of adenylyl cyclase in the WT membrane preparations that mimicked the intact cell heterologous desensitization; that is, there was an increase in the Kact for both epinephrine and prostaglandin E1 (PGE1) stimulations of adenylyl cyclase induced at the receptor level because neither forskolin- nor NaF-stimulated activity was affected. The desensitization was independent of agonist occupancy of the receptor, and the effects were blocked both by the active fragment (amino acids 5-22) of the specific inhibitor of cA.PK and by p[NH]ppA. cA.PKc treatment of kin- membranes resulted in a heterologous desensitization that resembled the effects of WT adenylyl cyclase, with the exception that forskolin-stimulated activity was also reproducibly decreased by 24%. cA.PKc had no effect on WT membranes isolated from cells that had previously undergone maximal heterologous desensitization during treatment with 10 microM forskolin. In contrast, cA.PKc-induced heterologous desensitization of kin- membranes was additive with the epinephrine-induced homologous desensitization of intact cells. Cell-free desensitizations were reversed by incubation of membranes with cA.PKc and ADP, conditions that drive the kinase reaction backward. The similarities of our cell-free cA.PKc-mediated heterologous desensitization of adenylyl cyclase with the intact cell desensitization support our hypothesis that heterologous desensitization of the WT lymphoma cells is mediated by cA.PK via a mechanism independent of homologous desensitization.

Receptor and effector interactions of G s Functional studies with antibodies to the α s carboxyl-terminal decapeptide

Antibodies generated to a synthetic decapeptide, RMHLRQYELL, representing the carboxyl-terminus of G s-α have been characterized in immunoblots and functional studies. This antibody, designated RM, reacts exclusively with a doublet of proteins of 52 and 45 kDa in immunoblots of bovine brain and wild-type S49 murine lymphoma cell membranes. No such reactivity is seen in membranes from cyc − S49 cells, which lack G s. RM blocks receptor-mediated activation of G s and adenylyl cyclase in membranes from wild-type S49 cells. RM could also immunoprecipitate adenylyl cyclase activity in detergent extracts from GTP[γ]S- or fluoride-preactivated bovine brain membranes; thus binding of α s to effector and carboxyl-terminal antibody was mutually compatible. Such experiments provide an approach for the elucidation of functionally relevant interactions of G-proteins with receptors and electors in the membrane.