Reconstitution of epidermal growth factor receptor transmodulation by platelet-derived growth factor in Chinese hamster ovary cells

Platelet-derived growth factor (PDGF) causes an acute decrease in the high affinity binding of epidermal growth factor (EGF) to cell surface receptors and an increase in the phosphorylation state of the EGF receptor at threonine654. The hypothesis that PDGF action to regulate the EGF receptor is mediated by the activation of protein kinase C and the subsequent phosphorylation of EGF receptor threonine654 was tested. The human receptors for PDGF and EGF were expressed in Chinese hamster ovary cells that lack expression of endogenous receptors for these growth factors. The heterologous regulation of the EGF receptor by PDGF was reconstituted in cells expressing [Thr654]EGF receptors or [Ala654]EGF receptors. PDGF action was also observed in phorbol ester down-regulated cells that lack detectable protein kinase C activity. Together these data indicate that neither protein kinase C nor the phosphorylation of EGF receptor threonine654 is required for the regulation of the apparent affinity of the EGF receptor by PDGF.     

Independent mechanisms account for the regulation by protein kinase C of the epidermal growth factor receptor affinity and tyrosine-protein kinase activity

The epidermal growth factor (EGF) receptor is a substrate for phosphorylation by the calcium- and phospholipid-dependent protein kinase (protein kinase C) at Thr654. The hypothesis that this phosphorylation is causally related to the regulation of the functional properties of the EGF receptor was tested by substitution of Thr654 with an alanine residue. Activation of protein kinase C using phorbol ester caused a decrease in the high affinity binding of EGF to Chinese hamster ovary cells expressing wild-type [Thr654]EGF receptors. Similar results were obtained with cells expressing mutated [Ala654]EGF receptors. The regulation of the protein kinase activity of the EGF receptor by protein kinase C was examined using a synthetic peptide substrate for tyrosine phosphorylation. Protein kinase C caused a Ca2+-dependent decrease in the tyrosine-protein kinase activity of the wild-type [Thr654]EGF receptor. In contrast, no inhibition of the tyrosine-protein kinase activity of the mutated [Ala654]EGF receptor caused by protein kinase C was detected. In further experiments, the desensitization of EGF action caused by the activation of protein kinase C was examined by investigating the regulation of the transferrin receptor by EGF. Phorbol ester was observed to cause the desensitization of signaling by the wild-type [Thr654] and mutated [Ala654]EGF receptors. These data are consistent with a role for the phosphorylation of EGF receptor Thr654 in the regulation of the receptor tyrosine-protein kinase activity. However, the inhibition of the high affinity binding of EGF to cell-surface receptors caused by protein kinase C does not require Thr654. It is concluded that independent mechanisms account for the regulation by protein kinase C of the EGF receptor affinity and tyrosine-protein kinase activity.     

Differential sensitivity of agonist- and antagonist-occupied gonadotropin-releasing hormone receptors to protein kinase C activators. A marker for receptor activation

Gonadotropin-releasing hormone (GnRH) stimulates release of pituitary gonadotropins by activating specific plasma membrane receptors. In the present studies, we have used activators of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) to probe the binding characteristics of agonist- or antagonist-occupied GnRH receptors in intact cell cultures, using a radioligand receptor assay. Specific binding of [125I-Tyr5,D-Ser(tBu)6,Pro9,NHEt]GnRH (Buserelin), a high-affinity GnRH agonist, was increased to 180% of control in the presence of 150 nM phorbol 12-myristate 13-acetate (PMA) or 100 nM phorbol 12,13-dibutyrate (PDB), and to 125% of control in the presence of 200 microM 1,2-dioctanoylglycerol, after 20 min at 23 degrees C. The PMA effects were associated with apparent increases in both binding affinity and number of binding sites. The effects of protein kinase C activators on Buserelin binding were concentration- and time-dependent and were not seen with 4 alpha-PMA or 1,2-dioctanoyl-3-Cl-glycerol, neither of which activate protein kinase C. In contrast, PMA had no measurable effects on specific binding of a GnRH receptor antagonist, Ac[D-pCl-Phe1,2,D-Trp3,125I-Tyr5,D-Lys6,D-Ala10]GnRH. When cell cultures were pretreated with 100 nM PDB in the absence of GnRH and then washed to remove the phorbol ester, no effects of prior protein kinase C activation were detected upon subsequent addition of Buserelin. However, when PDB pretreatment was carried out in the presence of 0.3 microM GnRH, residual enhancement of Buserelin binding, but not antagonist binding, was observed at either 23 or 4 degrees C. The radiolabeled agonist activated, and the antagonist blocked, GnRH receptor-mediated luteinizing hormone release and [3H]inositol phosphate production in cells preloaded with [3H]inositol. These findings suggest that the action of protein kinase C on the GnRH receptor, either direct or indirect, requires the receptor to be in an activated (agonist-occupied) state but does not require receptor internalization. The mechanism of these effects on GnRH agonist binding is not known but may involve sequestration of surface receptors, expression of new receptors, and/or modulation of GnRH receptor affinity.     

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.     

Mutation of Asn-391 within the conserved NPXXY motif of the cholecystokinin B receptor abolishes Gq protein activation without affecting its association with the receptor

Among the most conserved regions in the G-protein-coupled receptors is the (N/D)PX(2-3)Y motif of the seventh transmembrane domain (X represents any amino acid). The mutation of the Asn/Asp residue of this motif in different G-protein-coupled receptors was shown to affect the activation of either adenylyl cyclase or phospholipase C. We have mutated the Asn residue (Asn-391) of the NPXXY motif in the CCKBR to Ala and determined the effects of the mutation on binding, signaling, and G-proteins coupling after expression of the mutated receptor in COS cells. The mutated receptor displayed similar expression levels and high affinity CCK binding compared with the wild type CCKBR. However, unlike the wild type CCKBR, the mutated receptor was completely unable to mediate activation of either phospholipase C and protein kinase C-dependent and -independent mitogen-activated protein kinase pathways, indicating an essential role of Asn-391 in CCKBR signaling. Coimmunoprecipitation experiments allowed us to show that the inactive mutant retains an intact capacity to form stable complexes with G(q)alpha subunits in response to CCK. These results indicate that the formation of high affinity CCK-receptor-G(q) protein complexes is not sufficient to activate G(q) and suggest that Asn-391 is specifically involved in G(q) proteins activation.     

Activation of protein kinase C down-regulates IFN-gamma receptors

Treatment of mouse EL-4 cells with intracellular activators of protein kinase C, namely 4-phorbol 12-myristate 13-acetate (PMA) and diacylglycerol, resulted in 90% reduction in cell surface interferon-gamma (IFN-gamma) receptors as judged by iodinated-IFN-gamma binding. This did not seem to be due to a decreased in the receptor affinity, since that of the remaining surface receptors appeared to be significantly increased as shown in Scatchard plot analysis. Kinetics experiments revealed that a PMA treatment as short as 15 min was sufficient to induce a decrease of 30% of IFN-gamma receptors, whereas the highest levels of down-regulation were observed after 60-90 min. Treatment of EL-4 cells with calcium ionophore, A23187, although ineffective by itself, dramatically increased the ability of suboptimal PMA concentrations to mediate IFN-gamma receptor down-regulation. Finally, specificity studies revealed that PMA is particularly effective in decreasing the binding of IFN-gamma to T-lymphocytes. Altogether these results suggest a possible involvement of protein kinase C in the regulation of IFN-gamma receptor expression.     

Signals for activation and proliferation of murine T lymphocyte clones

Biochemical signals required for the growth of T cell clones were studied. Antigen-specific helper T cell clones, 6-1 and KO.6, could enter the state similar to the resting state where the cells expressed only small numbers of interleukin 2 (IL2) receptors and could not respond to IL2 without antigenic stimulation. A combination of a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and a calcium ionophore, A23187, induced the expression of IL2 receptors on resting 6-1 cells and induced DNA synthesis in the presence of IL2. TPA alone did not induce IL2 receptors. A23187 induced the expression of the receptors to some extent but did not induce DNA synthesis even in the presence of IL2. IL2 receptors induced by A23187 alone were mostly low affinity receptors, whereas the combination of TPA and A23187 induced high affinity receptors in addition to low affinity receptors. Resting KO.6 cells produced IL2 in response to a combination of TPA and A23187, whereas either one of the agents did not induce the production of IL2. Dicaprylin, a permeable diacylglycerol and a potent activator of protein kinase C (the Ca2+/phospholipid-dependent enzyme) could replace TPA in both cases when dicaprylin was repeatedly added to the culture. These results suggest that strong and continuous activation of protein kinase C together with calcium mobilization is required for IL2 production and IL2 receptor expression. On the contrary, signals for DNA synthesis generated by binding of IL2 to IL2 receptors are different from those for IL2 production and IL2 receptor expression, as the combination of TPA and A23187 could not induce DNA synthesis without IL2.     

Phorbol ester and interferon-gamma modulation of epidermal growth factor receptors on human amniotic (WISH) cells

In this study we report that pretreatment of human amniotic (WISH) cells with interferon gamma (IFN-gamma) in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the down-modulation of epidermal growth factor (EGF) receptors with respect to both receptor number and affinity. Scatchard analysis of EGF binding in the absence of both IFN-gamma and TPA indicated biphasic binding whereas addition of TPA resulted in the loss of the higher affinity class of sites. Pretreatment with IFN-gamma for 24 h enhanced the TPA-induced inhibition of EGF binding whereas IFN-gamma alone had no effect on binding. Protein kinase C (Ca2+/phospholipid-dependent enzyme) was examined as a possible mediator of IFN-induced EGF-receptor modulation; pretreatment of cells with IFN-gamma affected neither the binding of [3H]phorbol 12,13-dibutyrate to membrane or cytosolic fractions nor the protein kinase C activity, suggesting that IFN-gamma pretreatment did not result in translocation or activation of protein kinase C.     

Activation signals in human lymphocytes: interleukin 2 synthesis and expression of high affinity interleukin 2 receptors require differential signalling for the activation of protein kinase C

The mitogenic activation of resting T lymphocytes involves two distinct cellular events, the synthesis of the ultimate mitogen interleukin 2 and the synthesis and expression of receptors for it. In order to get more detailed information on the mechanisms associated with these activating steps (the effects of different stimuli, leading to activation of protein kinase C were investigated in human lymphocytes). The anti-T-cell receptor (TCR) and anti-CD3 monoclonal antibodies (BMA 031 and BMA 030, respectively), as well as the combination of the phorbol ester, TPA, with a calcium ionophore-induced interleukin 2 synthesis and subsequent proliferation in human peripheral blood lymphocytes. Incubation of cells with synthetic diacylglycerols and calcium ionophores proved to be effective in expression of high affinity interleukin receptors, no detectable amounts of interleukin 2 were, however, synthetized. When diacylglycerols were, however, added repetitively, interleukin 2 was also produced. Both anti-TCR/CD3 antibodies and TPA or DiC8 caused activation and translocation of protein kinase C from the cytosol to the plasma membrane. Significant differences, however, were observed between the time kinetics of the translocation of the enzyme. In plasma membranes of TPA-stimulated cells activation of protein kinase C was detectable up to 4 hr. In contrast, the highest specific activity of protein kinase C was measured in the plasma membranes after 15 min of DiC8 addition to cells. Anti-CD3 monoclonal antibodies activated protein kinase C in a biphasic manner. Shortly after binding of BMA 030 to the T cell antigen receptor/CD3 complex the activity of protein kinase C was increased in the plasma membrane, then it declined to control levels followed by a second long-lasting activation of the enzyme up to 4 hr. These results suggest different signal requirements for different activation steps. While for synthesis and expression of interleukin 2 receptors a short term activation of protein kinase C is sufficient, long-term activation of the enzyme is necessary for interleukin 2 synthesis in human lymphocytes.     

Heterologous regulation of EGF receptors in fibroblastic cells

Platelet-derived growth factor (PDGF) increases the mitogenic activity of epidermal growth factor (EGF) in several cells lines, including BALB/C-3T3. PDGF-treated BALB/C-3T3 cells manifest a reduced capacity to bind 125I-labeled EGF due to a loss of high affinity EGF receptors. Cholera toxin potentiates the ability of PDGF to both decrease EGF binding and initiate mitogenesis. Whether PDGF increases EGF sensitivity via its effects on EGF receptors is not known and requires a more complete understanding of the mechanism by which PDGF decreases EGF binding. 12-O-tetradecanoylphorbol 13-acetate (TPA) also reduces EGF binding in BALB/C-3T3 and other cells, presumably by activating protein kinase C and, consequently, inducing the phosphorylation of EGF receptors at threonine-654. PDGF indirectly activates protein kinase C, and EGF receptors in PDGF-treated WI-38 cells are phosphorylated at threonine-654. Thus, the effects of PDGF on EGF binding may also be mediated by protein kinase C. We investigated this hypothesis by comparing the actions of PDGF and TPA on EGF binding in density-arrested BALB/C-3T3 cells. Both PDGF and TPA caused a rapid, transient, cycloheximide-independent loss of 125I-EGF binding capacity. The actions of both agents were potentiated by cholera toxin. However, whereas TPA allowed EGF binding to recover, PDGF induced a secondary and cycloheximide-dependent loss of binding capacity. Most importantly, PDGF effectively reduced binding in cells refractory to TPA and devoid of detectable protein kinase C activity. These findings indicate that PDGF decreases EGF binding by a mechanism that involves protein synthesis and is distinct from that of TPA.     

Membrane-bound protein kinase C modulates receptor affinity and chemotactic responsiveness of Lewis lung carcinoma sublines to an elastin-derived peptide

The M27 and H59 variants of Lewis lung carcinoma differ in their responsiveness to the chemotactic elastin peptide Val-Gly-Val-Ala-Pro-Gly (VGVAPG). M27 cells, selected for metastasis to lung, are highly responsive to a positive gradient of VGVAPG. H59 cells, selected for metastasis to liver, do not migrate in response to VGVAPG. Although both cell types bind radiolabeled VGVAPG, Scatchard analysis of 125I-Tyr-VGVAPG binding reveals that M27 cells bind the chemoattractant with a Kd of 2.7 nM, whereas nonresponsive H59 cells bind the peptide with a Kd of 67 nM. These findings indicate that the failure of H59 cells to migrate in response to VGVAPG may be due to the reduced affinity of their VGVAPG receptors. Both receptor affinity and chemotactic responsiveness to VGVAPG can be modulated in each of these two tumor cell lines by the levels of active membrane-associated protein kinase C. Treatment of nonresponsive H59 cells with 12-O-tetradecanoylphorbol 13-acetate increases the level of membrane-bound protein kinase C activity with a concomitant increase in VGVAPG binding affinity and induction of chemotactic responsiveness to VGVAPG. Treatment of M27 cells with the protein kinase C inhibitor, staurosporine, reduces VGVAPG binding affinity and abrogates the chemotactic response. We conclude that chemotactic responsiveness of M27 and H59 tumor cells is dependent upon high VGVAPG receptor affinity, which is strongly correlated to high levels of membrane-bound protein kinase C activity.     

Regulation of platelet-activating factor receptor and PAF receptor-mediated arachidonic acid release by protein kinase C activation in rat Kupffer cells

Phorbol 12-myristate 13-acetate (PMA), a potent protein kinase C activator, caused down-regulation of receptors for platelet-activating factor (AGEPC) on the plasma membrane of rat Kupffer cells (40-50% reduction) but had a relatively minor effect on the binding affinity of the receptors for AGEPC (Kd = 0.30 nM vs 0.56 nM) when incubated with the cells for a short period of time (30-60 min). As a consequence, the AGEPC receptor-mediated arachidonic acid release was attenuated. The PMA-induced down-regulation of AGEPC receptors was concentration-dependent, specific, and transient (the maximal effect was observed at about 1 h and the level of specific [3H]AGEPC binding gradually returned to the control level within 8.5 h and even higher than the control level at 24 h after addition of PMA). Upon removing PMA from the culture medium, more than half of the lost receptors were replaced within 1 h at 37 degrees C and the recovery process appeared to be independent of protein synthesis. The ability of PMA to down-regulate the AGEPC receptors was lost in cells "down-regulated" for protein kinase C, suggesting that the receptor-regulatory effect of PMA is protein kinase C-dependent. Protein kinase C appeared to be involved in the AGEPC-induced arachidonic acid release since 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride, a protein kinase C inhibitor, attenuated the stimulatory effect of AGEPC in this system. In addition, AGEPC-induced [3H]arachidonic acid release was inhibited significantly in cells down-regulated for protein kinase C. The present study thus demonstrates that protein kinase C has dual actions in the regulation of AGEPC-mediated events, i.e., a positive forward action, regulating AGEPC-stimulated arachidonic acid release, and a negative action, which inactivates or down-regulates AGEPC receptors.     

Regulation of the epidermal growth factor receptor phosphorylation state by sphingosine in A431 human epidermoid carcinoma cells

The regulation of protein phosphorylation by sphingosine in A431 human epidermoid carcinoma cells was examined. Sphingosine is a competitive inhibitor of phorbol ester binding to protein kinase C (Ca2+/phospholipid-dependent enzyme) and potently inhibits phosphotransferase activity in vitro. Addition of sphingosine to intact A431 cells caused an inhibition of the phorbol ester-stimulated phosphorylation of two protein kinase C substrates, epidermal growth factor (EGF) receptor threonine 654 and transferrin receptor serine 24. We conclude that sphingosine inhibits the activity of protein kinase C in intact A431 cells. However, further experiments demonstrated that sphingosine-treatment of A431 cells resulted in the regulation of the EGF receptor by a mechanism that was independent of protein kinase C. First, sphingosine caused an increase in the threonine phosphorylation of the EGF receptor on a unique tryptic peptide. Second, sphingosine caused an increase in the affinity of the EGF receptor in A431 and in Chinese hamster ovary cells expressing wild-type (Thr654) and mutated (Ala654) EGF receptors. Sphingosine was also observed to cause an increase in the number of EGF-binding sites expressed at the surface of A431 cells. Examination of the time course of sphingosine action demonstrated that the effects on EGF binding were rapid (maximal at 2 mins) and were observed prior to the stimulation of receptor phosphorylation (maximal at 20 mins). We conclude that sphingosine is a potently bioactive molecule that modulates cellular functions by: 1) inhibiting protein kinase C; 2) stimulating a protein kinase C-independent pathway of protein phosphorylation; and 3) increasing the affinity and number of cell surface EGF receptors.     

Protein Kinase C Agonists Increase the Expression of Angiotensin II Receptors in Neuronal Cultures

Previous studies have shown that norepinephrine is important in the regulation of central angiotensin II receptors, an effect mediated by alpha 1-adrenergic receptors. Because alpha 1-adrenergic stimulation leads to inositol phospholipid hydrolysis and activation of protein kinase C, we have examined a possible role of this enzyme in the regulation of central angiotensin II (Ang II) receptors. In the present study, we have examined the effects of protein kinase C activators, phorbol esters, on the expression of Ang II receptors in neuronal cultures prepared from 1-day-old rat brains. The active phorbol ester phorbol-12-myristate-13-acetate (TPA) caused time- and concentration-dependent increases in the specific binding of [125I]Ang II to its receptors in neuronal cultures of normotensive and spontaneously hypertensive rat brains. The stimulatory effect of TPA on Ang II receptors was apparent within 15 min and reached a maximum between 1 and 2 h. Ang II specific binding had returned to control levels by 24 h. Various phorbol esters increased [125I]Ang II binding in accordance with their order of potency in stimulating protein kinase C activity. Saturation and Scatchard analysis revealed that the phorbol ester-induced increase in [125I]Ang II binding was due to an increase in the number of Ang II receptors. These observations indicate that activation of protein kinase C results in an increased expression of Ang II receptors in neuronal cultures from both normotensive and spontaneously hypertensive rat brains. The results suggest a possible role of phosphorylation in Ang II receptor expression in neuronal cultures.     

Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling

Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.     

Cloning of an apamin binding protein of vascular smooth muscle

The receptor for the bee venom derived neurotoxin, apamin, is widely believed to be an integral component of the small conductance calcium-activated potassium channel in many excitable cells. By affinity chromatography on immobilized apamin, a 78 kD apamin binding protein of the bovine brain synaptosomes was isolated. Antibodies were elicited against this protein and used to clone a cDNA from a porcine vascular smooth muscle expression library. This gene (Kcal 1.8) codes for a 438 amino protein with four potential transmembrane domains, one putative calcium binding site, a protein kinase C phosphorylation site, and a leucine zipper motif. Kcal 1.8 encoded protein has no significant sequence homologies with any known ion channels or receptors. Kcal 1.8 is likely to encode a protein associated with the small conductance calcium-activated potassium channel in vascular smooth muscle.     

EGF receptor affinity is regulated by intracellular calcium and protein kinase C

Phorbol esters specifically reduce the binding of epidermal growth factor to surface receptors in intact cells, but not when added directly to isolated membranes. We show that after treatment of intact cells with phorbol myristate acetate, 125I-EGF binding is reduced in membranes prepared subsequently. High-affinity binding of 125I-EGF is modulated by an intracellular calcium-dependent regulatory process. Preventing calcium entry with EGTA or enhancing intracellular calcium with A23187 in intact cells modulates EGF receptor affinity in membranes isolated subsequently. Also, EGTA attenuates the usual inhibition of EGF binding caused by phorbol esters. Membrane preparations do not respond to phorbol ester treatment because the calcium- and phospholipid-dependent protein kinase C is removed or inactivated during membrane isolation. Reconstitution of unresponsive membranes with purified C kinase alters phosphorylation of the EGF receptor and restores the inhibitory effect of phorbol esters on 125I-EGF binding previously observed only in intact cells. Thus, activation of the Ca++-dependent enzyme, C kinase, modulates EGF receptor affinity, possibly via altered receptor phosphorylation.     

Agonist-independent phosphorylation of purified cardiac muscarinic cholinergic receptors by protein kinase C

The results of several studies have suggested that muscarinic cholinergic receptors (mAChR) may be regulated by multiple pathways involving phosphorylation of the receptors. Previous studies have demonstrated that chick heart mAChR are phosphorylated by the beta-adrenergic receptor kinase (beta-AR kinase) in an agonist-dependent manner, and it has been suggested that this process may be linked to receptor desensitization. In this work, we present evidence that protein kinase C can phosphorylate the purified, reconstituted chick heart mAChR and can modify the interaction of the receptors with GTP binding proteins (G-proteins) that couple the receptors to effectors. Phosphorylation of the mAChR with protein kinase C occurred to an extent of approximately 5 mol of P/mol of receptor. Neither the rate nor the extent of the protein kinase C mediated phosphorylation of mAChR was agonist-dependent. Under the conditions tested, the initial rate of phosphorylation of the mAChR by protein kinase C was significantly more rapid than that obtained with the beta-AR kinase. At equilibrium, phosphorylation of mAChR by protein kinase C and beta-AR kinase was partially additive. The functional effects of protein kinase C mediated phosphorylation of the mAChR were assessed by comparing the abilities of purified G-proteins (Gi and Go) to reconstitute high-affinity agonist binding to phosphorylated and nonphosphorylated receptors. A significantly larger percentage of the receptors phosphorylated with protein kinase C exhibited G-protein-dependent high-affinity agonist binding, suggesting that phosphorylation of the receptors by protein kinase C modulates receptor function in a positive manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by platelet-derived growth factor in protein kinase C-deficient human fibroblasts

We have tested the hypothesis that the mechanism of platelet-derived growth factor (PDGF) and phorbol diester action to decrease the apparent affinity of the epidermal growth factor (EGF) receptor is the phosphorylation of the EGF receptor at the Ca2+/phospholipid-dependent protein kinase (protein kinase C) phosphorylation site, threonine 654. Protein kinase C-deficient cells were prepared by prolonged incubation of human fibroblasts with phorbol diester. Addition of phorbol diesters to these cells fails to regulate EGF receptor affinity or threonine 654 phosphorylation. In contrast, PDGF treatment of both control and protein kinase C-deficient fibroblasts causes a decrease in the apparent affinity of the EGF receptor and an increase in threonine 654 phosphorylation. Thus, the ability of PDGF or phorbol diester to modulate EGF receptor affinity occurs only when threonine 654 phosphorylation is increased. The stoichiometry of threonine 654 phosphorylation associated with a 50% decrease in the binding of 125I-EGF to high affinity sites was 0.15 versus 0.3 mol of phosphate per mole of EGF receptor when 32P-labeled fibroblasts are treated with PDGF or phorbol diester, respectively. It is concluded that EGF receptor phosphorylation at threonine 654 can be regulated by PDGF independently of protein kinase C, substoichiometric phosphorylation of the total EGF receptor pool at threonine 654 is caused by maximally effective concentrations of PDGF, and different extents of phosphorylation of EGF receptors at threonine 654 are observed for maximally effective concentrations of PDGF and phorbol diester, respectively. The data are consistent with the hypothesis that a specific subpopulation of EGF receptors that exhibit high affinity for EGF are regulated by threonine 654 phosphorylation.     

Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor

We examined the effect of insulin treatment on HTC cells transfected with large numbers of either normal insulin receptors (HTC-IR) or insulin receptors defective in tyrosine kinase (HTC-IR/M-1030). In both HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 microM) at 37 degrees C resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. In contrast, treatment with 10 nM insulin (20 h, 37 degrees C) also increased receptor affinity but had a smaller effect on the number of binding sites. 125I-Insulin binding to soluble receptors from HTC-IR and HTC-IR/M-1030 cells pretreated with insulin showed results similar to those obtained in intact cells. In both HTC-IR and HTC-IR/M-1030 cells, insulin enhanced insulin receptor degradation. In HTC-IR/M-1030 cells a 1-h incubation with insulin did not change receptor number and had only a small effect on receptor affinity; also there was no effect of insulin after a 20-h incubation at 15 degrees C. Inhibiting protein synthesis by pretreatment with cycloheximide (100 microM) did not block either the decrease in receptor number or the increase in receptor affinity. Both HTC-IR and HTC-IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells. These studies indicate, therefore, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor.