The processing of receptor-bound [125I-Tyr11]somatostatin by RINm5F insulinoma cells

The peptide somatostatin (SRIF) is secreted by delta cells of the endocrine pancreas and inhibits the secretion of insulin from pancreatic beta cells. We have previously shown that [125I-Tyr11]SRIF binds to specific, high affinity receptors on RINm5F insulinoma cells and that these receptors mediate the action of SRIF to inhibit insulin release. In the present study we investigated the processing of receptor-bound [125I-Tyr11]SRIF in this clonal cell line. Surface-bound and internalized peptides were distinguished by the ability of an acid/salt solution (0.2 M acetic acid, 0.5 M NaCl, pH 2.5) to dissociate only exposed ligand-receptor complexes. Surprisingly, greater than 80% of saturably bound [125I-Tyr11]SRIF was removed by this acid wash independent of the time or temperature of the binding incubation. In contrast, the processing of receptor-bound [125I]EGF (epidermal growth factor) in RINm5F cells was markedly temperature-dependent. Although over 90% of saturably bound [125I]EGF was dissociated by acid after a 4 degrees C binding incubation, less than 10% was removed by acid treatment after 37 degrees C binding. The radioactivity released upon dissociation of receptor-bound [125I-Tyr11]SRIF was analyzed by high performance liquid chromatography and shown to consist of a mixture of intact peptide (40%) and [125I]tyrosine (60%). However, neither the rate of [125I-Tyr11]SRIF dissociation nor its degradation were affected by NH4Cl, methylamine, or leupeptin at concentrations which inhibited the lysosomal degradation of [125I] EGF. Of 11 other protease inhibitors tested, only the metalloendoprotease inhibitor, phosphoramidon, substantially reduced the degradation of receptor-bound [125I-Tyr11]SRIF. These data indicate that, unlike [125I] EGF, receptor-bound [125I-Tyr11]SRIF is not rapidly internalized by RINm5F cells and is degraded by a nonlysosomal process which may involve a metalloendoprotease.     

Characterization of bombesin receptors in a rat pituitary cell line

Bombesin is a tetradecapeptide which stimulates prolactin secretion in rats and man and in cultures of GH4C1 cells, a clonal strain of rat pituitary tumor cells. We have utilized [125I-Tyr4]bombesin to identify and characterize specific high affinity receptors in GH4C1 cells. Scatchard analysis of equilibrium binding data at 4 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (RT = 3600 +/- 500 sites/cell). The value for the equilibrium dissociation constant (Kd = 1.2 +/- 0.4 nM) agreed closely with the ED50 (0.5 nM) for bombesin stimulation of prolactin release. [125I-Tyr4]Bombesin binding at steady state at 37 degrees C was inhibited by increasing concentrations of unlabeled bombesin in a dose-dependent manner, with an ID50 = 1.4 +/- 0.2 nM. However, binding of [125I-Tyr4] bombesin was not inhibited by 100 nM thyrotropin-releasing hormone, vasoactive intestinal peptide, epidermal growth factor, or somatostatin. Therefore, [125I-Tyr4]bombesin binds to a receptor distinct from the receptors for other peptides which regulate hormone secretion by GH4C1 cells. The analog specificity for high affinity binding showed that the receptors for bombesin recognize the COOH-terminal octapeptide sequence in the molecule. Among five pituitary cell strains tested, two which contained saturable binding sites for [125I-Tyr4]bombesin (GH4C1 and GH3) had previously been shown to respond to bombesin with increased hormone secretion, whereas three which lacked receptors (GC, F4C1, and AtT20/D16v) were unresponsive. Therefore, the [125I-Tyr4]bombesin binding sites appear to be necessary for the biological actions of bombesin. Examination of the processing and metabolism of receptor-bound peptide demonstrated that at 4 degrees C [125I-Tyr4]bombesin binds to receptors on the surface of GH4C1 cells. At 37 degrees C, receptor-bound peptide is rapidly internalized and subsequently degraded in lysosomes. In summary, we have characterized for the first time specific, high affinity pituitary bombesin receptors which are necessary for the biological action of bombesin.     

Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line.

Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thyrotropin releasing hormone action in pituitary cells. Protein kinase C-mediated effects on the epidermal growth factor receptor

Thyrotropin releasing hormone (TRH) causes phosphatidylinositol bisphosphate hydrolysis to form inositol trisphosphate and diacylglycerol. Since diacylglycerol activates protein kinase C (Ca2+/phospholipid-dependent enzyme), this enzyme may be involved in mediating the physiological response to TRH. Activation of protein kinase C leads to phosphorylation of receptors for epidermal growth factor (EGF) and decreased EGF affinity. The present study examined the effect of TRH on EGF binding to intact GH4C1 rat pituitary tumor cells to test whether TRH activates protein kinase C. Cells were incubated with TRH at 37 degrees C and specific 125I-EGF binding was then measured at 4 degrees C. 125I-EGF binding was decreased by a 10-min treatment with 0.1-100 nM TRH to 30-40% of control in a dose-dependent manner. 125I-EGF binding was not altered if cells were incubated at 4 degrees C, although TRH receptors were saturated or in a variant pituitary cell line without TRH receptors. TRH (10 min at 37 degrees C) decreased EGF receptor affinity but caused little change in receptor density, 125I-EGF internalization, or degradation. When cells were incubated continuously with TRH, there was a recovery of 125I-EGF binding after 24 h. Incubation with the protein kinase C activating phorbol ester TPA caused an immediate (less than 10 min) profound (greater than 85%) decrease in 125I-EGF binding followed by partial recovery at 24 h. Maximally effective doses of TRH and TPA decreased EGF receptor affinity with half-times of 3 min. EGF treatment (5 min) caused an increase in the tyrosine phosphate content of several proteins; prior incubation with TRH resulted in a small decline in the EGF response. GH4C1 cells were incubated with 500 nM TPA for 24 h in order to down-regulate protein kinase C. Protein kinase C depletion was confirmed by immunoblots and the effects of TRH and TPA on 125I-EGF binding were tested. TRH and TPA were both much less effective in cells pretreated with phorbol esters. TRH increased cytoplasmic pH measured with an intracellularly trapped pH sensitive dye after mild acidification with nigericin. This TRH response is presumed to be the result of protein kinase C-mediated activation of the amiloride-sensitive Na+/H+ exchanger and was blunted in protein kinase C-depleted cells. All of these results are consistent with the view that TRH acts rapidly in the intact cell to activate protein kinase C and that a consequence of this activation is EGF receptor phosphorylation and Na+/H+ exchanger activation.     

Inhibition of the binding of 125I-labelled epidermal growth factor to mouse cells by a mitogen in goat mammary secretions.

Pre-colostrum and colostrum from goats cause a marked inhibition of the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The ability of these secretions to inhibit 125I-EGF binding is closely correlated with the ability to stimulate DNA synthesis in quiescent 3T3 cell cultures, suggesting that goat mammary secretions may contain an EGF-related mitogen. However, the material in colostrum which inhibits 125I-EGF binding to Swiss 3T3 cells is a basic protein with Mr greater than 20000 and is thus quite different from mouse and human EGF. Furthermore, the colostral-mediated inhibition of 125I-EGF binding, although rapid and apparently competitive, differs from the inhibition of binding induced by native, unlabelled EGF. Thus, the inhibitory effect of colostrum is markedly decreased when the assay temperature is shifted from 37 degrees C to 4 degrees C whereas unlabelled EGF is an effective competitive inhibitor at both 37 degrees C and 4 degrees C. Incubation of cells with EGF causes a reduction in cell surface EGF receptors whereas exposure to colostrum does not induce down-regulation of the EGF receptor. Our results suggest that the colostral factor does not bind directly to EGF receptors but inhibits 125I-EGF binding by an indirect mechanism which involves a temperature-sensitive step.     

Recycling of epidermal growth factor in A431 cells

The fate of epidermal growth factor (EGF) after internalization by A431 cells was studied. First, cells containing 125I-EGF-receptor complexes in endosomes were obtained. Subsequent incubation of the cells at 37 degrees C resulted in the recycling of 125I-EGF from endosomes to the cell surface in the receptor-bound state and the gradual release of recycled ligand into the medium. The excess of unlabeled EGF blocked both rebinding and re-internalization of recycled 125I-EGF to produce enhanced accumulation of ligand in the medium. The rate of recycling was shown to be much higher than that of EGF degradation.     

Interactions between the receptors for platelet-derived growth factor and epidermal growth factor

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.     

Alteration of somatostatin but not growth hormone-releasing factor pituitary binding sites in obese Zucker rats.

The present study was designed to determine whether the diminution of growth hormone (GH) secretion that occurs in obese Zucker rats is related to alterations of GH-releasing factor (GRF) or somatostatin (SRIF) pituitary binding sites. Cold saturation studies were performed in pituitary homogenates of 4-month-old lean and obese rats, using [125I-Tyr10]hGRF(1-44)NH2 as radioligand and [127I-Tyr10]hGRF-(1-44)NH2 as competitor, and in pituitary membrane preparations, using [125I-Tyr0, D-Trp8]SRIF14 as radioligand and [127I-Tyr0, D-Trp8]SRIF14 as competitor. In lean rats, analysis of the curves by the Ligand program revealed the presence of two distinct classes of GRF binding sites, the first being of high affinity (0.74 +/- 0.11 nM) and low capacity (118 +/- 31 fmol/mg protein), the second being of lower affinity (880 +/- 240 nM) and higher capacity (140 +/- 35 pmol/mg protein), and of a single class of SRIF binding sites (affinity: 0.40 +/- 0.12 nM; capacity: 24 +/- 6 fmol/mg protein). In obese rats, no difference was observed in GRF binding parameters for both classes of sites, but the concentration of somatostatin binding sites was reduced by 67% when compared to their lean littermates. These findings suggest that the SRIF pituitary receptors are down-regulated in obese Zucker rats and indicate that no alteration of GRF pituitary binding sites contribute to the blunted GH secretion observed in this model of obesity.     

Diacylglycerol treatment rapidly decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells

The synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol (OAG) and phorbol esters activate protein kinase C in intact cells. We report here that OAG inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The inhibition was detected as early as 1 min after treatment at 37 degrees C and persisted for at least 120 min. The effect of OAG was reversed upon removal of this diacylglycerol. Detailed Scatchard analysis of 125I-EGF binding to Swiss 3T3 cells at 4 degrees C after a 1 h incubation with a saturating dose of OAG at 37 degrees C, demonstrates that this OAG pretreatment does not change the apparent number of EGF receptors but causes a marked decrease in their apparent affinity for the ligand. Prolonged treatment (40 h) of the cells with phorbol dibutyrate (PBt2) which causes a marked decrease in the number of phorbol ester binding sites and in the activity of protein kinase C, prevented the inhibition of 125I-EGF binding by both PBt2 and OAG. The results support the possibility that protein kinase C plays a role in the transmodulation of the EGF receptor in intact cells.     

Identification of somatostatin receptors by covalent labeling with a novel photoreactive somatostatin analog

We have synthesized two photoreactive derivatives of somatostatin, namely [125I-Tyr11,azidonitrobenzoyl (ANB)-Lys4]somatostatin and [125I-Tyr11,ANB-Lys9]somatostatin, and used them to characterize somatostatin receptors biochemically in several cell types. Saturation binding experiments carried out in the dark demonstrated that [125I-Tyr11,ANB-Lys4]somatostatin bound with high affinity (KD = 126 +/- 39 pM) to a single class of binding sites in GH4C1 pituitary cell membranes. The affinity of this analog was similar to that of the unsubstituted peptide [125I-Tyr11]somatostatin (207 +/- 3 pM). In contrast, specific binding was not observed with [125I-Tyr11,ANB-Lys9]somatostatin. The binding of both [125I-Tyr11,ANB-Lys4]somatostatin and [125I-Tyr11]somatostatin was potently inhibited by somatostatin (EC50 = 300 pM) whereas at 100 nM unrelated peptides had no effect. Furthermore, both pertussis toxin treatment and guanyl-5'yl imidophosphate (Gpp(NH)p) markedly reduced [125I-Tyr11,ANB-Lys4]somatostatin binding. Thus, [125I-Tyr11,ANB-Lys4]somatostatin binds to G-protein coupled somatostatin receptors with high affinity. To characterize these receptors biochemically, GH4C1 cell membranes were irradiated with ultraviolet light following the binding incubation, and the labeled proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A major band of 85 kDa was specifically labeled with [125I-Tyr11,ANB-Lys4]somatostatin but not with [125I-Tyr11,ANB-Lys9]somatostatin or [125I-Tyr11]somatostatin. The binding affinity of the 85-kDa protein for [125I-Tyr11,ANB-Lys4]somatostatin was very high (Kd = 34 pM). Labeling of this protein was inhibited competitively by somatostatin (EC50 = 140 +/- 80 pM) but not by unrelated peptides. Furthermore, this band was not labeled in pertussis toxin-treated membranes or in untreated membranes incubated with Gpp(NH)p. Finally, [125I-Tyr11,ANB-Lys4]somatostatin specifically labeled bands of 82, 75, and 72 kDa in membranes prepared from mouse pituitary AtT-20 cells, rat pancreatic acinar AR4-2J cells, and HIT hamster islet cells, respectively. Thus, [125I-Tyr11,ANB-Lys4]somatostatin represents the first photolabile somatostatin analog able to bind to receptors with high affinity. Our studies demonstrate that this novel peptide covalently labels specific somatostatin receptors in a variety of target cell types.     

Recycling of epidermal growth factor-receptor complexes in A431 cells: identification of dual pathways

The intracellular sorting of EGF-receptor complexes (EGF-RC) has been studied in human epidermoid carcinoma A431 cells. Recycling of EGF was found to occur rapidly after internalization at 37 degrees C. The initial rate of EGF recycling was reduced at 18 degrees C. A significant pool of internalized EGF was incapable of recycling at 18 degrees C but began to recycle when cells were warmed to 37 degrees C. The relative rate of EGF outflow at 37 degrees C from cells exposed to an 18 degrees C temperature block was slower (t1/2 approximately 20 min) than the rate from cells not exposed to a temperature block (t1/2 approximately 5-7 min). These data suggest that there might be both short- and long-time cycles of EGF recycling in A431 cells. Examination of the intracellular EGF-RC dissociation and dynamics of short- and long-time recycling indicated that EGF recycled as EGF-RC. Moreover, EGF receptors that were covalently labeled with a photoactivatable derivative of 125I-EGF recycled via the long-time pathway at a rate similar to that of 125I-EGF. Since EGF-RC degradation was also blocked at 18 degrees C, we propose that sorting to the lysosomal and long-time recycling pathway may occur after a highly temperature-sensitive step, presumably in the late endosomes.     

Insulin-like growth factor 1 and insulin reduce epidermal growth factor binding to Swiss 3T3 cells by an indirect mechanism that is apparently independent of protein kinase C

Insulin-like growth factor 1 and insulin reduced the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells by 15-20% at 37 degrees C, but not at 4 degrees C. Scatchard analysis indicated that IGF-1 and insulin affected the higher-affinity component of EGF binding, an effect previously associated with the activation of protein kinase C. However, the inhibition of 125I-EGF binding by IGF-1 and insulin was increased, not reduced, when the cells were treated with high concentrations of phorbol esters to down-modulate protein kinase C. We suggest that IGF-1 and insulin activate a protein kinase with similar or overlapping specificity to that of protein kinase C.     

Characterization of the rebinding of 125I-epidermal growth factor released from BALB/c-3T3 cells following accumulation in the presence of chloroquine

Lysosomotropic amines, such as chloroquine and methylamine, increase the intracellular accumulation of 125I-EGF by inhibiting lysosomal degradation. It has been shown previously that BALB/c-3T3 cells, prelabeled at 4 degrees C with 125I-EGF for 3 h and subsequently chased at 37 degrees C in the presence of chloroquine, internalized the surface bound 125I-EGF which was subsequently released into the extracellular medium in a high molecular weight form which co-migrated with native 125I-EGF. The secreted 125I-EGF rebound to the cells from which it was released more efficiently than does peptide in the extracellular media. We now show that when the BALB/c-3T3 cells were prelabeled at 37 degrees C for 2 h in the presence of chloroquine, the internalized 125I-EGF released into the medium was in a high molecular weight form which co-migrated with native 125I-EGF and did not rebind anymore efficiently than did peptide in the extracellular media. This lack of rebinding was not due to an alteration in the 125I-EGF molecule since it was still capable of rebinding to naive A431 cells, nor was it due to the exhaustion of EGF receptors on the BALB/c-3T3 cells. The inhibition of rebinding was observed only when the cells were treated with EGF in the presence of chloroquine, and was not due to a general down-regulation of membrane receptors. The differences between the rebinding of 125I-EGF at 4 degrees C and 37 degrees C suggest that EGF may be processed via different pathways in the cell.     

Regulation of thyroidal inducibility of Na,K-ATPase and binding of epidermal growth factor in wild-type and cold-sensitive E1a mutant type 5 adenovirus-transformed CREF cells

We have analyzed the relationship between expression of the transformed phenotype and thyroid hormone (triiodothyronine, T3) inducibility of Na,K-ATPase and binding of 125I-epidermal growth factor (EGF) to cell membrane receptors in wild-type (wt) and mutant type 5 adenovirus (Ad5)-transformed CREF cells displaying a cold-sensitive (cs) expression of the transformed phenotype. CREF cells respond to thyroid hormone treatment with increased Na,K-ATPase activity and bind similar levels of 125I-EGF at 32 degrees C, 37 degrees C and 39.5 degrees C. In contrast, CREF cells transformed by wt Ad5 or the E1a plus E1b-transforming genes of wt Ad5 are refractile to T3 treatment and bind lower levels of 125I-EGF than CREF cells at all three temperatures. By employing a series of cloned CREF cell lines transformed by a host-range cold-sensitive mutant virus, H5hr1 or H5dl101, or the E1a or E1a plus E1b genes from these viruses, we have investigated expression of the transformed state and its relationship with hormone inducibility and EGF binding. When cs virus, cs E1a- or cs E1a plus E1b-transformed CREF clones were grown at 32 degrees C, a nonpermissive transforming temperature in which cs-transformed cells exhibit properties similar to untransformed CREF cells, T3 induced Na,K-ATPase activity and these cells bound similar levels of 125I-EGF as CREF cells. However, when cs virus- and cs Ela plus E1b-transformed CREF clones were incubated at 37 degrees C or 39.5 degrees C, temperatures at which cs-transformed cells exhibit properties similar to wt Ad5-transformed CREF cells, they did not respond to T3 and bound lower levels of 125I-EGF than CREF cells. In the case of cs E1a-transformed CREF clones, thyroid hormone responsiveness was observed at both 32 degrees C and 37 degrees C, but not at 39.5 degrees C. By performing temperature shift experiments--i.e. 32 degrees C to 37 degrees C, 32 degrees C to 39.5 degrees C, 37 degrees C to 32 degrees C, and 39.5 degrees C to 32 degrees C, it was demonstrated that after a shift from lower to higher temperature a 24-hr lag period was required for cs-transformed CREF cells to lose T3 inducibility and exhibit reduced EGF binding, whereas 96 hr after a shift from higher to lower temperature a 96-hr lag period was required for cs-transformed cells to regain T3 inducibility and increased 125I-EGF binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of functional receptors for somatostatin in rat pituitary cells in culture.

GH4C1 cells are a clonal strain of rat pituitary tumor cells which synthesize and secrete prolactin and growth hormone. Somatostatin, a hypothalamic tetradecapeptide, inhibits the release of growth hormone and, under certain circumstances, also prolactin from normal pituitary cells. We have prepared [125I-Tyr1]somatostatin (approximately 2200 C1/mmol) and have shown that this ligand binds to a limited number of high affinity sites on GH4C1 cells. Half-maximal binding of somatostatin occurred at a concentration of 6 x 10(-10) M. A maximum of 0.11 pmol of [125I-Tyr1]somatostatin was bound per mg of cell protein, equivalent to 13,000 receptor sites per cell. The rate constant for binding (kon) was 8 x 10(7) M(-1) min(-1). The rate constant for dissociation (koff) was determined by direct measurement to be 0.02 min(-1) both in the presence and absence of excess nonradioactive somatostatin. Binding of [125I-Tyr1]somatostatin was not inhibited by 10(-7) M thyrotropin-releasing hormones. Substance P, neurotensin, luteinizing hormone-releasing hormone, calcitonin, adrenocorticotropin, or insulin. Of seven nonpituitary cell lines tested, none had specific receptors for somatostatin. Somatostatin was shown to inhibit prolactin and growth hormone production by CH4C1 cells. The dose-response characteristics for binding and the biological actions of somatostatin were essentially coincident. Furthermore, among several clonal pituitary cell strains tested, only those which had receptors for somatostatin showed a biological response to the hormone. We conclude that the characterized somatostatin receptor is necessary for the biological actions of somatostatin on GH4C1 cells.     

The effect of the lysosomotropic agent primaquine on the endocytosis of the epidermal growth factor receptors in A431 cells]

It has been shown elsewhere that the epidermal growth factor (EGF) in A431 cells can recycle in receptor-bound state (Teslenko et al., 1987; Sorkin et al., 1989, 1991). Present study deals with the action of primaquine, a lysosomotropic agent, on EGF-receptor complexes (EGF-RC). By the method of indirect immunofluorescence with anti-EGF-R monoclonal antibody it is found that following a 1 h incubation of cells at 37 degrees C in the presence of EGF a bright staining of endosomes appears in the intranuclear region, while after incubation of the cells at 4 degrees only margins of cells are stained. Such a pattern of fluorescence is peculiar of endocytosis in A431 cells. When the cells were incubated in the presence of a 0.3 mM primaquine for 1 h, the immunostaining is changed: bright compact spot in the para-Golgi region appeared. The effect of primaquine is reversible. When the cells after preincubation with EGF were incubated in the absence of EGF for 3 h at 37 degrees C, the staining of cell margins could be observed again, demonstrating the recycling of EGF-RC. Under similar conditions of cell incubation, but in the presence of primaquine, the staining of the para-Golgi region was not changed. In the experiments with 125I-EGF it was shown that intracellular accumulations of 125I-EGF were maintained when the cells were incubated in the presence of 0.3 mM primaquine. It is concluded that primaquine inhibits the recycling of EGF-R in A431 cells.     

High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes.

The binding of the radiolabelled bombesin analogue [125I-Tyr4]bombesin to guinea-pig lung membranes was investigated. Binding of [125I-Tyr4]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (Bmax = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (KD = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [Tyr4]bombesin, neuromedin B and neuromedin C inhibited the binding of [125I-Tyr4]bombesin in an order of potencies as follows: [Tyr4]bombesin greater than bombesin greater than or equal to neuromedin C much greater than neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.     

Protamine enhances epidermal growth factor (EGF)-stimulated mitogenesis by increasing cell surface EGF receptor number. Implications for existence of cryptic EGF receptors

Treatment of Swiss mouse 3T3 cells and human epidermoid carcinoma A431 cells with protamine at 37 degrees C increased the 125I-epidermal growth factor (EGF) binding activity at 4 degrees C. The effect of protamine on the increase of 125I-EGF binding activity appeared to be time, temperature, and dose dependent. This up-modulation of 125I-EGF binding by protamine correlated with protamine enhancement of EGF-stimulated mitogenesis, with respect to the magnitude of the effect and the dose response curves. Scatchard plot analyses indicated that protamine induced an increase in numbers of both high and low affinity EGF receptors without affecting their affinities. Protamine also increased functionally active EGF receptors in plasma membranes and solubilized membranes. This was evidenced by Scatchard plot analyses and by a protamine-induced increase of 125I-EGF-EGF receptor complex and an increase in EGF-stimulated phosphorylation of the EGF receptor. Combined with column chromatography of the solubilized EGF receptor on protamine-agarose gel, these results suggest that protamine may increase the EGF receptor number by directly activating cryptic EGF receptors in the plasma membrane.     

The hepatic binding and uptake kinetics of epidermal growth factor: studies with isolated rat hepatocytes

Hepatocytes are known to bind and internalize a variety of small molecular weight proteins by a process known as receptor-mediated endocytosis (RME). The purpose of this investigation was to characterize the binding and uptake kinetics of a small protein known to be taken up by the liver by RME, epidermal growth factor (EGF), using suspensions of freshly isolated rat hepatocytes. Rat hepatocytes accumulated 125I-EGF (90 pM) in a temperature-dependent fashion. Isolated hepatocytes incubated at 37 degrees C with 125I-EGF began to release a TCA-soluble radiolabeled material into the incubation medium with a lag period of 20 min. EGF uptake by isolated hepatocytes was linear for only 60 seconds and displayed saturation kinetics (apparent Km of 4 nM and a Vmax of 105 fM/min/10(6) cells). Hepatocytes incubated at 4 degrees C bound, but did not internalize, EGF. Under these conditions, EGF binding was saturable at concentrations above 8 nM. A Scatchard analysis revealed that the average number of receptors per hepatocyte was 7.7 X 10(4) with a dissociation constant of 2.6 nM. These data demonstrate that freshly isolated hepatocytes are capable of binding, internalizing and metabolizing EGF and thus are a good model to study RME of small molecular weight proteins.     

Somatostatin pretreatment increases the number of somatostatin receptors in GH4C1 pituitary cells and does not reduce cellular responsiveness to somatostatin

The GH4C1 pituitary cell line contains specific plasma membrane receptors for the inhibitory neuropeptide somatostatin (SRIF). Unlike other peptides which bind to cell surface receptors on these cells, SRIF is not rapidly internalized via receptor-mediated endocytosis. Here we examined the effects of chronic SRIF pretreatment on the subsequent ability of GH4C1 cells to bind and respond to this hormone. Treatment of cells with 100 nM SRIF increased [125I-Tyr1]SRIF binding to a maximum value of 220% of control after 20 h. Scatchard analysis demonstrated that the number, but not the affinity, of the receptors was altered. The effect of SRIF was dose-dependent (ED50 = 2.3 +/- 0.4 nM), was not mimicked by an inactive analog, and was specific for the SRIF receptor. Furthermore, pretreatment of cells with other agents, which mimic SRIF's action to decrease intracellular cAMP and free Ca2+ concentrations, did not mimic the SRIF-induced increase in receptor number. Thus, occupancy of the SRIF receptor was required for SRIF receptor up-regulation. Inhibition of protein synthesis with cycloheximide did not prevent the SRIF-induced increase in receptors, consistent with an effect of SRIF to either reduce receptor degradation or cause slow redistribution of preexisting receptors to the plasma membrane. In contrast to the effects on receptor binding, pretreating cells with SRIF did not alter either basal cAMP levels or the potency of SRIF to inhibit cAMP accumulation (ED50 = 0.5 +/- 0.2 nM). However, the maximum cAMP produced by stimulators of adenylyl cyclase was increased. The observation that chronic SRIF exposure did not cause homologous desensitization in GH4C1 cells and increased rather than decreased SRIF receptor number is consistent with the fact that this neuropeptide is not rapidly internalized by receptor-mediated endocytosis.