Number Of Membrane Receptors Research Articles

Simultaneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines

We investigated whether the folate receptor α-isoform (FRα), which is overexpressed on ovarian carcinoma cells, is functionally active in internalizing the physiological form of folate, 5-methyl tetrahydrofolate (THF). Six ovarian tumor cell lines, expressing different levels of FRα (COR ≫ OVCAR3 > IGROV1 > OVCAR4 > SKOV3 > OVCAR5), were maintained in folate-depleted medium and internalization of 10 nM evaluated as acid-resistant radioactivity at 0° and 37°C. The amount of 5-methyl[3H]THF present in this fraction was not strictly related to the number of membrane receptors, since even cell lines with low FRα expression, e.g., OVCAR4, showed efficient internalization. Time-course studies indicated that, whereas no uptake was detected at 0°C, at 37°C the internalized fraction showed a slow and constant increase, until 4 h. At this time, the internalized radioactivity represented <50% of the total bound in COR, OVCAR3 and IGROV1 cells, whereas the other cell lines tested internalized fourfold more folate than their surface binding capacity. The incubation in the presence of a concentration (50 nM) of 5-methyl[3H]THF, which best ensures receptors saturation on cells with highest FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, the increase of the uptake was more pronounced, particularly in SKOV3 cells. These results, together with the accumulation curves of folic acid (FA) and 5-methylTHF at 37°C, suggested the presence of a molecule on ovarian carcinoma cells with high affinity for reduced folates, possibly a reduced folate carrier (RFC). Measurement of radioactivity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5-methyl[3H]THF was translocated to the cytosol and, despite differences in membrane levels of FRα expression this internalized fraction was similar in both cell lines. Inhibition experiments to selectively block FRα or RFC activity showed a differential sensitivity of the two pathways depending on the cell line examined. Internalization was more consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FRα activity, e.g., incubation with excess FA and phosphatidylinositol specific phospholipase C, whereas Probenecid, which preferentially inhibits the carrier-mediated pathway, showed a strong inhibitory effect on both cell lines. These findings suggest that the internalization of 5-methylTHF in these tumor cells depends not only on the level of overexpressed FRα, but another transport route, with features characteristic for RFC, is functional and participates in folate uptake. J. Cell. Biochem. 65:479–491. © 1997 Wiley-Liss Inc.

Simultaneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines

We investigated whether the folate receptor alpha-isoform (FR alpha), which is overexpressed on ovarian carcinoma cells, is functionally active in internalizing the physiological form et folate, 5-methyl tetrahydrofolate (THF). Six ovarian tumor cell lines, expressing different levels of FR alpha (COR > > OVCAR3 > IGROV1 > OVCAR4 > SKOV3 > OVCAR5), were maintained in folate-depleted medium and internalization of 10 nM evaluated as acid-resistant radioactivity at 0 degree and 37 degrees C. The amount of 5-methyl[1H]THF present in this fraction was not strictly related to the number of membrane receptors, since even cell lines with low FR alpha expression, e.g., OVCAR4, showed efficient internalization. Time-course studies indicated that, whereas no uptake was detected at 0 degree C, at 37 degrees C the internalized fraction showed a slow and constant increase, until 4 h. At this time the internalized radioactivity represented < 50% of the total bound in COR, OVCAR3 and IGROV1 cells, whereas the other cell lines tested internalized fourfold more folate than their surface binding capacity. The incubation in the presence of a concentration (50 nM) of 5-methyl[3H]THF, which best ensures receptors saturation on cells with highest FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, the increase of the uptake was more pronounced, particularly in SKOV3 cells. These results, together with the accumulation curves of folic acid (FA) and 5-methylTHF at 37 degrees C, suggested the presence of a molecule on ovarian carcinoma cells with high affinity for reduced folates, possibly a reduced folate carrier (RFC). Measurement of radioactivity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5-methyl[3H]THF was translocated to the cytosol and, despite differences in membrane levels of FR alpha expression this internalized fraction was similar in both cell lines. Inhibition experiments to selectively block FR alpha or RFC activity showed a differential sensitivity of the two pathways depending on the cell line examined. Internalization was more consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FR alpha activity, e.g., incubation with excess FA and phosphatidylinositol specific phospholipase C, whereas Probenecid, which preferentially inhibits the carrier-mediated pathway, showed a strong inhibitory effect on both cell lines. These findings suggest that the internalization of 5-methylTHF in these tumor cells depends not only on the level of overexpressed FR alpha, but another transport route, with features characteristic for RFC, is functional and participates in folate uptake.

G protein coupled receptors as modules of interacting proteins: a family meeting.

The emergence on earth of the first true cell would not have been possible without the presence of molecules ensuring exchanges of information between this membrane-limited, metabolically organized system and its surrounding environment. The function of message recognition and transmission inside the cell is allotted to receptors, most of which are embedded in the plasma membrane and allow hydrophilic compounds to act as extracellular messengers. The evolutionary success of these transmission systems is strikingly evidenced by the tremendous number of membrane receptors which have been described to date in all the organisms from bacteria to mammals. Incidentally, receptors are among the molecules of utmost interest for the pharmacologist, since in most cases a compound must bind a receptor to be biologically active (best stated by Ehrlich’s famous aphorism “Corpora non agunt nisifixata” [Ehrlich, 1906]).

Differential role of the carboxyl-terminal tyrosine in down-regulation and sequestration of the m2 muscarinic acetylcholine receptor.

Muscarinic acetylcholine receptor (mAChR) number can be altered in response to sustained agonist exposure. Short term agonist exposure (seconds to minutes) causes a rapid removal of mAChR from the cell surface (sequestration) while agonist exposure for longer periods of time (hours) causes a decrease in total receptor number (down-regulation). Tyrosine residues located in the cytoplasmic tails of a number of membrane receptors have been demonstrated to be important in the regulation by either sequestration, as is the case with the mannose 6-phosphate receptor and other receptors endocytosed via clathrin coated vesicles, or down-regulation, as is the case with the beta 2-adrenergic receptor. Mutation of the lone cytoplasmic tail tyrosine residue (Tyr-459) of the mammalian m2 mAChR to Phe, Trp, or Ala did not affect agonist-induced sequestration, although it significantly attenuated agonist-induced down-regulation. Conversion of m2 Tyr-459 to Ile did not affect the rate or extent of agonist-induced sequestration or down-regulation, but the sensitivity of this mutant receptor to agonist-induced down-regulation was slightly decreased. Agonist and antagonist binding as well as functional coupling to the inhibition of cAMP accumulation was unaffected by any of the mutations to Tyr-459. These results are the first to identify a site in a mAChR involved in the down-regulation of receptor in response to agonist.

Bombesin treatment enhances vasopressin receptors in Swiss 3T3 cells

Cells possess receptors for multiple different peptides that regulate a wide spectrum of biological processes. Although examples of homologous and heterologous downregulation have been reported, relatively little is known about the interaction between different peptides in modulating cellular activities. Here we demonstrate that pretreatment of Swiss 3T3 fibroblasts with 10 nM bombesin for 48 h enhanced the 45Ca2+ efflux acutely stimulated by vasopressin. The effect was not reciprocal, since preincubation with vasopressin did not affect the bombesin-stimulated Ca2+ efflux. Measurement of displaceable [3H] vasopressin binding demonstrated that bombesin pretreatment increases the hormonal binding by 3.8 +/- 0.2-fold (SE; n = 14) measured at 37 degrees C or at 4 degrees C. Scatchard analysis at 4 degrees C indicated that the increased binding reflects an increase in the number of vasopressin receptors without any significant effect on the apparent affinity of binding. Furthermore, addition of cycloheximide completely prevented the increase in [3H] vasopressin binding induced by bombesin. We conclude that long-term bombesin pretreatment induces heterologous enhancement of vasopressin responsiveness by increasing the number of membrane receptors.

Fibrozytenreaktion auf Interleukin-1-Stimulation bei Keratokonus

The reduced mechanical strength of the tissues around a keratoconus and the ultrastructural disorganization of its matrix components are caused by proteolytic breakdown and incomplete repair. In most biochemical studies on this subject, reduced collagen content and increased collagenase and gelatinase activity have been reported. Since interleukin-1 (IL-1) promotes secretion of prostaglandin E-2 (PG-E2 and collagenase, we were interested in the number of membrane receptors for these cytokines on keratoconus fibroblasts and determined the corresponding dissociation constant (kD). We also studied the kinetics of cyclo-oxygenase and the synthesis of PG-E2, IL-1 and collagen. We compared the data with data from normal human corneas. 1) There was a significant difference (P < 0.05) between the specific binding of [125I]-IL-1 in keratoconic and normal corneal cells. In keratoconus, the number of receptors was four times higher (7000 +/- 100 binding sites per cell) than in normal corneas (1540 +/- 100 binding sites per cell). However, there was little difference between the dissociation constants for keratoconic ([6.21 +/- 0.5] x 10(-11) and normal ([6.27 +/- 0.5] x 10(-11) corneal cells. 2) Under the study conditions, interleukin-1 was found neither in normal nor in keratoconic cornea. 3) Synthesis of PG-E2 without stimulation of cells by IL-1, which was observed in 8 corneas with and 8 without keratoconus, showed that keratoconus cells secrete ten times more PG-E2 than normal corneals cells. If IL-1 is added to the incubation medium, which contains [3H]-labeled arachidonic acid, PG-E2 synthesis increases more than in normal corneal cells. 4) After adding IL-1, the collagen concentration in keratoconus cells in culture was more pronounced than in normal corneal cells. It is still not known why the anomalies discussed above occur in keratoconus. The increase in IL-1 receptor expression and the high level of cyclooxygenase activity are very probably due to genetic causes, because they are observed even after 40 generations of cell cultures.

Membrane function and vascular reactivity

This communication examines the possibility that nitric oxide (NO) production by endothelial cells results from changes in cell membrane fluidity. Lysophosphatidylcholine (LPC) alters fluidity of the endothelial cell membranes causing vascular relaxation. Through membrane alterations LPC influences function of a number of membrane receptors and modulates enzyme activity. As a result of detergent action, lysophosphatidylcholine (LPC) causes activation of guanylate cyclase, stimulates sialyltransferase and regulates protein kinase C activity. It has already been demonstrated that ionic detergents, such as Triton X-100 also cause vascular relaxation, possibly induced by NO production from endothelial cells. It is postulated that production of nitric oxide results from changes in membrane viscosity; this may represent a mechanism for its regulation in biological systems.

Differential calcium mobilization by vasopressin, angiotensin II, gastrin-releasing peptide, and adenosine triphosphate in adult and fetal hepatocytes. Relevance for the activation of calcium-dependent enzymes

Early signals elicited after membrane receptor binding of agonists, the transmembrane signaling pathway of which involves activation of phosphoinositide-specific phospholipase C, were compared in fetal (22 days gestation) and adult rat hepatocytes. Free cytosolic calcium changes varied depending on the agonist and type of stimulated cells. Angiotensin II and ATP elicited the maximal responses in both types of cells, whereas the maximal Ca2+ increase produced by vasopressin was twice as much in adult than in fetal hepatocytes. The opposite response was observed for bombesin- or gastrin-releasing peptide-stimulated cells. Triggering of fetal and adult hepatocytes with substances that maximally promote endoplasmic reticulum calcium release or phosphoinositide-specific phospholipase C activation revealed that at least for the actions mediated through the angiotensin II and P2 purinergic receptor, the agonist stimulation was near the maximal response capacity of the signaling pathway. Agreement was observed between the relative number of membrane receptors and the biological responses.

Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor.

Basic fibroblast growth factor, a potent angiogenesis inducer, stimulates urokinase (uPA) production by vascular endothelial cells. In both basic fibroblast growth factor-stimulated and -nonstimulated bovine capillary endothelial and human umbilical vein endothelial cells single-chain uPA binding is mediated by a membrane protein with a Mr of 42,000. Exposure of bovine capillary or endothelial human umbilical vein endothelial cells to pmolar concentrations of basic fibroblast growth factor results in a dose-dependent, protein synthesis-dependent increase in the number of membrane receptors for uPA (19,500-187,000) and in a parallel decrease in their affinity (KD = 0.144-0.790 nM). With both cells, single-chain uPA binding is competed by synthetic peptides whose sequence corresponds to the receptor-binding sequence in the NH2-terminal domain of uPA. Exposure of bovine capillary endothelial cells to transforming growth factor beta 1, which inhibits uPA production and upregulates type 1 plasminogen activator inhibitor, the major endothelial cell plasminogen activator inhibitor, has no effect on uPA receptor levels. These results show that basic fibroblast growth factor, besides stimulating uPA production by vascular endothelial cells, also increases the production of receptors, which modulates their capacity to focalize this enzyme on the cell surface. This effect may be important in the degradative processes that occur during angiogenesis.

The “cellular state”: The way to regain specificity and diversity in hormone action

In contrast with the high specificity achieved in the effects of hormones and growth factors by their interaction with a large number of membrane receptors, a loss of information seems to take place due to the small number of second messenger systems. To retain specificity one has to consider the cellular state as defined in a state machine by a pattern of activity. Different molecular mechanisms are considered as possible candidates to establish such states following the initial ligand-receptor interaction and the activation of one or several second messengers.

Fibronectin biosynthesis: influence on fibroblast adhesion during chick embryo development

Eight d (8d) and 16d (16d) chick embryo fibroblasts (CEF) exhibited marked differences in their adhesive capacity on plastic support, but not on fibronectin substratum. This suggests differences in fibronectin (FN) expression and/or FN receptor expression. Both 8d and 16d CEF expressed an identical number of membrane receptors for FN with similar affinity. In contrast, the newly synthesized FN appeared de novo in 30 min in 8d CEF versus 60 min in 16d CEF. This difference is not due to a modification of the polypeptide chain biosynthetic rate. The FN synthesized in 8d CEF became insensitive to endo beta-N-acetyl-glucosaminidase H (endo H) treatment after 20 min, whereas it remained sensitive to endo H until 60 min in 16d CEF. Post-translational modifications of N-linked mannose-rich chains to complex type chain may account for the difference in the expression of cell surface FN and thus for the difference in cell adhesion capacity to plastic.

Dietary fat-mediated changes in hepatic apoprotein [formula omitted] receptor in the guinea pig: Effect of polyunsaturated, monounsaturated, and saturated fat

These studies investigated the effects of dietary fat quality on guinea pig plasma total and low-density lipoprotein (LDL) cholesterol and triglyceride levels; LDL composition, density, and binding affinity to hepatic apolipoprotein (apo) B E receptors; and hepatic apo B E receptor levels. Animals were fed semi-synthetic diets containing 7.5% (weight per weight) fat—either corn oil (CO), olive oil (OL), or lard. Plasma cholesterol levels were significantly lower on the CO diet compared with the OL and lard diets. The isolated LDL had mean peak densities that ranged from 1.071 for LDL from lard-fed animals to 1.075 for LDL from CO- and OL-fed animals. The cholesterol ester to protein ratio of the LDL particle decreased in CO-fed guinea pigs. Binding studies showed that these compositional changes of the LDL had no effect on the binding affinity of the particles to a standardized hepatic membrane preparation. Membrane phospholipid fatty acid compositions were significantly different among the three dietary fat groups. When hepatic membranes were incubated with 125I-labeled LDL, the receptor-mediated binding of LDL to membranes from CO-fed animals increased 1.5-fold compared with binding to membranes from OL- and lard-fed guinea pigs. Scatchard plots indicated an increase of 50% in receptor number in membranes of animals fed the CO diet, whereas the affinity of the hepatic apo B E receptor for LDL (K d) was virtually identical for all membranes. It is concluded from these data that the increase in specific binding observed in hepatic membranes from CO-fed guinea pigs is caused by an increased number of LDL receptors and not an increased affinity for the LDL particle.

Enhancement of adenosine A2 and prostaglandin E1 receptor-mediated cAMP generation by prior exposure of Swiss 3T3 fibroblasts to Ca2+-mobilizing receptor agonists or phorbol ester: Activation of protein kinase C triggers increases in the receptor density in cell membranes

Production of cAMP in response to adenosine A2 or prostaglandin E1 receptor stimulation was, but the production induced by a beta-adrenergic agonist or forskolin was not, enhanced by prior exposure of Swiss 3T3 fibroblasts to agonists of Ca2+-mobilizing receptors or phorbol ester for 3 h. The enhancement reflected potentiation of the receptor-coupled activation of adenylate cyclase and the 2-fold increase in the adenosine A2 receptor number in membranes under these conditions. No enhancement was observed, however, when the medium used for the prior exposure was further supplemented with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or staurosporin, inhibitors of protein kinase C, neither of which affected the cAMP responses of the nonexposed cells. It is very likely, therefore, that activation of protein kinase C triggers the increase in certain receptor density in membranes, thereby enhancing the receptor-coupled cAMP-generating responses. The physiological significance of such cross-talk between cellular signaling systems is discussed in comparison with similar previous observations.

Monoclonal antibodies against Vero cells that protect against diphtheria toxin

B. J. Rönnberg, B. C. Lidgerding and J. L. Middlebrook. Monoclonal antibodies against Vero cells that protect against diphtheria toxin. Toxicon 27, 1095–1104, 1989.—Mice were immunized with a cell line (Vero) that possesses a high number of membrane receptors for diphtheria toxin. Spleen cells from these mice were fused with SP2/0-Ag14 cells and two cell lines (1A2 and 2D2) isolated by screening for the ability of their secreted antibodies to inhibit binding of radiolabeled diphtheria toxin to Vero cells. These antibodies protected Vero cells from the inhibition of protein synthesis mediated by diphtheria toxin. The antibodies were purified, iodinated, and their binding characteristics investigated. At 4°C, the association of 1A2 and 2D2 with Vero cells was saturable ( K D ≈ 10 −8 M) and indicated about 10 6 binding sites/cell. Diphtheria toxin did not inhibit the binding of either radiolabeled antibody. Monoclonal antibody 1A2 completely inhibited 125I-2D2 binding and vice versa. Trypsin or phospholipase C treatment of Vero cells had no effect on the ability of the monoclonal antibodies to bind to the cells. These findings suggest that: (1) the two monoclonal antibodies recognize the same or closely related epitopes and (2) the antibodies bind a domain distinct from the toxin binding site or to a subcomponent of the diphtheria toxin receptor that is present at many other cell surface sites. These antibodies offer a powerful tool to study the structure, processing and mode of action of diphtheria toxin receptors.

Isoproterenol response following transfection of the mouse beta 2-adrenergic receptor gene into Y1 cells.

The beta 2-adrenergic receptor (beta 2AR) gene was isolated from a mouse genomic library, sequenced and shown to share 93% identity with the hamster beta 2AR cDNA at the amino acid level. This mouse beta 2AR genomic clone was transfected into the Y1 mouse adrenal cortex tumor cell line. Northern blot and S1 nuclease analysis showed that the beta 2AR-transfected cells expressed an mRNA of the appropriate size to encode the receptor. Membrane receptor number and affinities for various beta-adrenergic agonists demonstrated that the transfected clone encoded a beta 2AR protein product. Incubation of the transfected Y1 cells, which do not normally possess beta 2AR, with the beta 2AR agonist, isoproterenol, resulted in an increase in the rate of steroid secretion by these cells as well as a rapid change in cell morphology. This response was fully blocked by the beta 2AR antagonist, propranolol. Prolonged incubation of the cells with isoproterenol resulted in agonist insensitivity and an 80% reduction in membrane receptor number.

Association of decreased myocardial beta-receptors and chronotropic response to isoproterenol and exercise in pigs following chronic dynamic exercise.

The effects of chronic dynamic exercise on myocardial beta-adrenergic and muscarinic cholinergic receptors and chronotropic sensitivity to isoproterenol were studied in 5 Yucatan miniswine. Right atrial and left ventricular biopsies, heart rate responses to isoproterenol, and maximal exercise treadmill testing were obtained before and after 10-19 weeks of treadmill running. Radioligand studies using 125I-iodocyanopindolol (ICYP) and 3H-quinuclidinyl benzilate (QNB) were used to determine the number of beta-adrenergic and muscarinic cholinergic receptors. Maximal oxygen consumption increased from 52 +/- 5 to 65 +/- 7 ml/kg/min (mean +/- SD; p less than 0.02), maximal workload from 530 +/- 111 to 1,074 +/- 179 KPM/min (p less than 0.01), resting heart rate decreased from 91 +/- 13 to 62 +/- 4 beats/min (p less than 0.01), heart rate at 75% of pretraining maximal workload decreased from 253 +/- 15 to 196 +/- 12 beats/min (p less than 0.01), and maximal exercise heart rate decreased from 273 +/- 6 to 254 +/- 9 beats/min (p less than 0.01). Decreased heart rate responsiveness to adrenergic stimulation was observed following chronic exercise. Maximal isoproterenol-stimulated heart rate decreased from 225 +/- 13 to 185 +/- 28 beats/min (p less than 0.05) and the slope of the isoproterenol dose-response relation decreased from 63 +/- 16 to 40 +/- 16 (p less than 0.05). Radioligand studies revealed a decrease in beta-receptor number in the right atrium following chronic exercise (61 +/- 9 vs. 34 +/- 8 fmol/mg; p less than 0.02), but receptor number in membranes from the left ventricle did not change (60 +/- 9 vs. 62 +/- 4 fmol/mg).(ABSTRACT TRUNCATED AT 250 WORDS)

INHIBITION OF FATTY ACID OXIDATION BY SULFONYLUREAS

Sulfonylureas potentiate the cellular action of insulin, not by changing membrane receptor number as previously thought, but by unknown postbinding mechanisms. We investigated the direct effects of tolazamide on fatty acid oxidation in isolated intact liver mitochondria, prepared from fed and fasted rats. Total ketone body formation and CO2 production (x10% of total oxidative products) were measured using 1−14C palmitate. Ketogenesis results are expressed in nmols/min/mg prot. (± SEM).

Effect of age on nuclear triiodothyronine receptors in circulating human lymphocytes

Increasing age in both animals and man has been associated with various alterations in thyroid hormone economy. This study reports no difference in either serum T3, serum T4, or lymphocyte membrane receptor number or affinity in healthy men of increasing age.

Alloxan-induced diabetes reduces beta-adrenergic receptor number without affecting adenylate cyclase in rat ventricular membranes.

We have investigated alterations in beta-adrenergic receptors and adenylate cyclase activity in myocardial membranes from normal and alloxan-treated diabetic rats. Saturation curves of [3H]dihydroalprenolol binding yielded a Bmax of 96.3 +/- 3.9 fmol/mg protein in normal membranes and 47.6 +/- 3.9 fmol/mg protein in diabetic membranes. Decreased receptor number in membranes from diabetic animals was not accompanied by alteration in receptor affinity for either antagonists or agonists to the beta-receptor. We were unable to detect any alteration in adenylate cyclase activity in similar ventricular membranes. Adenylate cyclase activity in the basal state or in the presence of sodium fluoride, guanyl-5'-yl imidodiphosphate, or isoproterenol, with or without GTP, was not altered by the alloxan-induced diabetic state. Stimulation of adenylate cyclase activity by forskolin, the novel diterpene activator, also was not altered by diabetes. The results suggest that while diabetes reduced beta-receptor number, this is not reflected in any other component of the adenylate cyclase complex.

Monoclonal antibodies against receptor for epidermal growth factor induce early and delayed effects of epidermal growth factor.

Mice were immunized with human epidermoid carcinoma cells (A-431 cell line) that possess an unusually high number of membrane receptors for epidermal growth factor (EGF). Spleen cells from these mice were fused with NSI cells, a nonsecreting murine myeloma. The immunoglobulins secreted by the obtained hybridomas were screened for specific binding to A-431 cells and selected according to their ability to inhibit the binding of radiolabeled EGF to the membrane of A-431 cells. Several antibodies secreted by cloned hybrid lines were found to inhibit the binding of radiolabeled EGF to membrane receptors of living A-431 cells, human foreskin fibroblasts, and mouse 3T3 fibroblasts and also to membrane preparations from A-431 cells. These monoclonal antibodies induced the early and delayed biological effects mediated by EGF. Like EGF, the antibodies induced morphological changes in A-431 cells and enhanced the phosphorylation of endogenous membrane proteins in membranes from these cells. They also stimulated DNA synthesis in human foreskin fibroblasts. These observations support the notion that the biological information of the EGF-receptor complex resides in the membrane receptor. Furthermore, the antibodies offer a powerful tool to study the structure, processing, and mode of action of EGF receptors.