Molecular cloning of human macrophage capping protein cDNA. A unique member of the gelsolin/villin family expressed primarily in macrophages.

Macrophage capping protein (MCP) is a Ca(2+)-sensitive protein which reversibly blocks the barbed ends of actin filaments but does not sever preformed actin filaments. The human cDNA for MCP has been cloned and sequenced. The derived amino acid sequence predicts a polypeptide of 38.4 kDa. Human MCP expressed in Escherichia coli using a pET12a vector was functionally identical to the native protein purified from rabbit alveolar macrophages with respect to Ca2+ sensitivity and ability to block monomer exchange at the barbed end of actin filaments. Sequence comparison with other actin-binding protein sequences indicates that MCP is a member of the gelsolin/villin family of barbed end blocking proteins. Unlike gelsolin, this protein has a limited tissue distribution being detected primarily in macrophages where it was abundant, representing 0.9-1% of the total cytoplasmic protein. Northern blot analysis of U937 and HL60 cells differentiated to macrophage-like cells demonstrated that MCP message increases to 2.6 and greater than 7 times initial levels, respectively. Human MCP displays a 93% amino acid sequence identity with two recently described mouse proteins, gCap39 and Mbh1. Its abundance in macrophages and the corresponding increases in mRNA levels upon promyelocyte and monocyte development into macrophages indicate that MCP may play an important role in macrophage function.     

Nullipotent Teratocarcinoma Cells Acquire the Pluripotency for Differentiation by Fusion with Somatic Cells

Abstract. By fusion of nullipotent embryonal carcinoma F9 cells with certain somatic cells freshly collected from differentiated tissues such as thymus and lens, pluripotent hybrid cell lines were obtained. They exhibited a wide spectrum of differentiation, including neural tubes, cartilages, skeletal muscles, ciliated epithelia and others, in solid tumors formed after injection into syngeneic mice. Cells from these tumors differentiated into several cell types when cultured in vitro. A possibility of the introduction of genes to code the factors for regulating differentiation into F9 cells by fusion is suggested.     

The cytosolic glycoprotein FP21 of Dictyostelium discoideum is encoded by two genes resulting in a polymorphism at a single amino acid position

FP21 is a glycoprotein within the cytosolic compartment of Dictyostelium which carries an unusual carbohydrate modification(s) including the sugars fucose, galactose and N-acetylglucosamine. The soluble pool of FP21 from crude extracts resolves chromatographically into two fractions that differ in their glycosylation. Previous gene-mapping studies indicating the existence of two loci suggested that the FP21 fractions might be encoded by different genes. To address this issue, the two genes were cloned and sequenced, leading to the prediction that the protein products would differ by only a single amino acid, Ser or Ala, at codon 39. Protein sequence data on CNBr fragments of purified FP21 showed that both gene products are found in both fractions of the soluble pool. After further purification, the two fractions were no longer chromatographically resolvable, and there was no evidence for charge heterogeneity as determined by 2-D gel electrophoresis of whole cells. Thus, the initial separation of the different soluble subpopulations of this protein appears to be due to distinct molecular complexes, possibly related to differential glycosylation, and is not the result of the genetically-encoded amino acid polymorphism.     

Expression of various viral and cellular enhancer-promoters during differentiation of human embryonal carcinoma cells

Alterations in the pattern of gene expression were studied during differentiation of the human embryonal carcinoma (EC) cell line NEC14. NEC14 cells can be induced to differentiate by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA). The efficiency of DNA transfection of undifferentiated and differentiated NEC14 cells was compared by measuring the activities of endogenous and exogenously introduced promoters for the beta-actin gene and heat shock protein 70 gene. The results indicated that the efficiency was not significantly different in cells of these two states. Under the conditions used, all the viral enhancer-promoters tested showed very little or no activity in undifferentiated cells, but activities of SV40, BKV, adenovirus and RSV enhancers were greatly increased after differentiation. Activities of these viral enhancers in differentiated cells were completely repressed by cotransfection with the adenovirus E1A gene. An E1A-inducible promoter of the adenovirus E2 gene showed stronger activity in differentiated than in undifferentiated cells, and was not activated efficiently by cotransfection with the E1A gene in either undifferentiated or differentiated cells. These results indicate that factor(s) regulating activities of various enhancer-promoters in NEC14 cells is or are different from E1A-like factor(s) present in mouse EC F9 cells.     

Cloning and characterization of mouse cullin4B/E3 ubiquitin ligase

Heat induced differentiation of mouse embryonal carcinoma cells PCC4 has been reported earlier. We have further characterized the phenotype of the differentiated cells and by DD-RT-PCR identified several partial cDNAs that are differentially expressed during differentiation. Nucleotide homology search revealed that the genes corresponding to some of the up-regulated partial cDNAs are indeed part of differentiation pathway. 5′ extension of an EST that has homology to one of the partial cDNAs led to the identification of mouse cullin4B. Cullin4B is coded by a separate gene and has a unique and longer amino-terminal end with a putative nuclear localization signal sequence (NLS). We have cloned, expressed and raised antibodies against the amino and carboxy-terminal halves of cullin4B. Immuno staining of differentiated PCC4 cells with N-terminal Cul4B antibody showed enhanced expression of Cul4B and its translocation into the nucleus upon differentiation. Transient transfection of a chimeric gene encoding the N-terminal part of Cul4B fused to green fluorescent protein into PCC4 cells revealed that the protein was localized in the nucleus confirming the functional significance of the putative NLS. Since cullins are involved in recognition of specific proteins for degradation, based on the evidence presented here, we hypothesize that cullin4B is probably involved in differentiation specific degradation/ modification of nuclear proteins.     

The N-terminal region of the medium subunit (PduD) packages adenosylcobalamin-dependent diol dehydratase (PduCDE) into the Pdu microcompartment

Salmonella enterica produces a proteinaceous microcompartment for B(12)-dependent 1,2-propanediol utilization (Pdu MCP). The Pdu MCP consists of catabolic enzymes encased within a protein shell, and its function is to sequester propionaldehyde, a toxic intermediate of 1,2-propanediol degradation. We report here that a short N-terminal region of the medium subunit (PduD) is required for packaging the coenzyme B(12)-dependent diol dehydratase (PduCDE) into the lumen of the Pdu MCP. Analysis of soluble cell extracts and purified MCPs by Western blotting showed that the PduD subunit mediated packaging of itself and other subunits of diol dehydratase (PduC and PduE) into the Pdu MCP. Deletion of 35 amino acids from the N terminus of PduD significantly impaired the packaging of PduCDE with minimal effects on its enzyme activity. Western blotting showed that fusing the 18 N-terminal amino acids of PduD to green fluorescent protein or glutathione S-transferase resulted in the association of these fusion proteins with the MCP. Immunoprecipitation tests indicated that the fusion proteins were encapsulated inside the MCP shell.     

Interaction of guanine-nucleotide-binding regulatory proteins with chemotactic peptide receptors in differentiated human leukemic HL-60 cells.

Human leukemic HL-60 cells were differentiated into neutrophil-like cells by treatment with dimethylsulfoxide (Me2SO) or N6,O2'-dibutyryladenosine 3',5'-phosphate (Bt2cAMP), and membrane fractions were prepared from the differentiated cells. Receptors for fMLF (fM,N-formylmethionine) and guanine-nucleotide-binding regulatory proteins (G proteins) serving as the substrate for pertussis toxin (islet-activating protein; IAP) were extracted from cell membranes then reconstituted into phospholipid vesicles. The binding of fMLF to the reconstituted vesicles (or the membranes) was determined with 10 nM [3H] fMLF. In both cases, high-affinity binding to vesicle preparations from the Me2SO- and Bt2cAMP-induced cells was abolished following treatment with IAP, suggesting that fMLF receptors were functionally coupled to IAP-sensitive G proteins in each of the two vesicle types. However, the high-affinity fMLF binding was much higher in vesicle preparations originating from Bt2cAMP-induced cells than in those from Me2SO-induced cells, although the amount of IAP-substrate G protein reconstituted into the each phospholipid vesicles preparation was not significantly different from the other. The G proteins of the two differentiated cells were both identified as inhibitory forms (Gi-2) based on their electrophoretic mobilities and immunoblot analyses. When purified Gi-2 from rat brain was reconstituted into the two IAP-treated vesicles, high-affinity fMLF binding was restored in a similar manner in both. IAP-substrate G proteins partially purified from the two differentiated HL-60 cells were also effective in restoring high-affinity fMLF binding to the IAP-treated vesicles. However, a significant difference was observed that the reconstituted binding was higher with the G-protein-rich fraction from Bt2cAMP-induced cells than with that from Me2SO-induced cells, with each of the two IAP-treated vesicle types. These results suggest that the different high-affinity binding of fMLF observed in the two differentiated HL-60 cells are due to a difference in the property of endogenous G proteins rather than fMLF receptors, though the two G proteins are indistinguishable from each other in terms of the subtype of G protein, Gi-2.     

Regulation of mouse mammary tumor viral RNA synthesis in embryonal carcinoma cells and in teratocarcinoma derived myoblasts.

The expression of the mouse mammary tumor virus (MMTV) was studied in undifferentiated embryonal carcinoma cells (ECC), in partially differentiated myoblastic cells derived from ECC cells, and in fully differentiated myotubes. Whereas no appreciable amount of MMTV RNA could be detected in embryonal carcinoma cells, hybridation with radioactive viral cDNA revealed relatively large quantities of tumor virus RNA in the teratocarcinoma derived myoblasts. The MMTV RNA level was strongly reduced after differentiation of myoblasts into myotubes. The glucocorticoid hormone dexamethasone which stimulates the MMTV RNA synthesis in differentiated mammary cells did not affect this synthesis in myoblastic cells. By contrast, the apparently repressed synthesis of MMTV RNA in myotubes was almost completely overcomed with dexamethasone.     

A novel brain-specific mRNA encoding nuclear protein (necdin) expressed in neurally differentiated embryonal carcinoma cells

A novel DNA sequence has been isolated from a subtraction cDNA library of P19 embryonal carcinoma cells treated with retinoic acid which induces neural differentiation of the stem cells. The cDNA insert (4B) hybridized with a single 1.7 kb mRNA, whose abundance was markedly increased in P19 cells after retinoic acid treatment. The 1.7 kb mRNA was also expressed in the brain, but not in other non-neuronal tissues. A 1.6 kb cDNA insert (4BFL), which was cloned by screening another cDNA library with the 4B probe, encodes a novel protein sequence of 325 amino acids (Mr 36,831). The protein expressed in 4BFL-transfected COS cells was translocated into the nuclei as detected with antibodies against subsequences of the predicted protein. The antibodies stained the nuclei of neurally differentiated P19 cells but not of the undifferentiated stem cells. This novel mRNA encoding the nuclear protein, termed necdin, may represent a useful marker for the differentiation and development of brain cells.     

The amount of a specific cellular protein (p53) is a correlate of differentiation in embryonal carcinoma cells

A specific cellular protein of molecular weight of 53–55,000 (p53) has been shown to be induced in all SV40 transformed cells. A similar protein has also been shown to be present in embryonal carcinoma cells and in midgestation murine embryo primary cells, which are not infected by SV40. In embryo cell primaries the amount of the protein was shown to decrease with the increase in the stage of embryo development. As differentiation or decrease in cell growth rate can account for this, and since the growth rate of embryo primary cells cannot be measured, we chose to investigate various embryonal carcinoma cells. We report that the p53 is present in a pluripotent embryonal carcinoma cell OTT6050, and in its differentiated parietal endoderm derivative, PYS-2 cells. The amount of p53 is higher in the undifferentiated EC stem cells than in the differentiated PYS-2 (parietal endoderm) cells. The amount of the protein decreases in F9 embryonal carcinoma cells induced to differentiate to a parietal endoderm cell type by treatment with retinoic acid, as it does following spontaneous differentiation of OTT6050 EC cells. To determine if a change in growth rate, rather than differentiation, might acount for the diminished levels of this protein, the amount ofp53 was measured in growing and in growth arrested cell populations. When the growth rate of F9 cells was reduced by treatment with 8-bromocyclic AMP there was no change in the amount of p53. The half life of the p53 was compared in the undifferentiated and the differentiated cell types to determine if a change in stability might account, in part, for the altered levels of this protein. The p53 is found to be most stable in the SV40 transformed established clonal cells. It is less stable in the fibroblast clonal cells which were not transformed by SV40. The results of these experiments indicate that a decrease in the amount of p53 primarily correlates with differentiation in the embryonal carcinoma cell lines studied and not with cell growth rate. Furthermore, the decrease appears to be related (in part) to the decreased stability of the p53.     

DNA cloning and amino acid sequence determination of a major constituent protein of mammalian nucleoli

Using a cDNA probe encoding the nucleolar protein N038 of Xenopus laevis, we have isolated clones that code for the corresponding mammalian protein from cDNA libraries of mouse embryonal carcinoma and fetal liver cells. The murine cDNA-derived amino acid sequence defines a polypeptide of 292 amino acids (including the initial methionine) of a total molecular weight of 32560 and identifies a single 1.5 kb mRNA on Northern blot hybridization. This polypeptide, which is highly homologous to the Xenopus protein N038, displays an organization in three major domains: (1) an aminoterminal portion of 119 amino acids, which shows a striking homology to nucleoplasmin of Xenopus; (2) a central portion of 68 amino acids that contains two extended acidic domains, a shorter of 13 residues and a longer of 29 residues, separated by an interval enriched in positively charged amino acids; (3) a carboxyterminal portion of 105 amino acids, which is almost identical to the reported partial amino acid sequence of human and rat nucleolar protein termed B23. The sequence comparisons show that the murine protein is the mammalian counterpart to the nucleolar protein N038 of Xenopus and is compatible with the idea that both proteins N038 represent the amphibian and murine equivalents to the human and rat nucleolar phosphoprotein B23. Special sequence features and predicted conformations of this protein are discussed in relation to the specific localization and the possible functions of this major nucleolar protein.     

Molecular cloning of cDNAs for two subunits of rat multicatalytic proteinase. Existence of N-terminal conserved and C-terminal diverged sequences among subunits

cDNA clones for two subunits (designated subunits K and L) of rat liver multicatalytic proteinase (MCP) were isolated using oligonucleotide probes synthesized according to their partial amino acid sequences. The encoded polypeptides of subunits K and L consisted of 255 and 261 amino acid residues with calculated molecular mass of 28.3 kDa and 29.5 kDa, respectively. Northern blot analysis revealed that subunits K and L were expressed in all tissues examined and their expression patterns were almost identical. The deduced amino acid sequences showed no similarities to known protein sequences other than the recently reported sequences of rat and Drosophila MCP subunits. Sequence comparison of MCP subunits of rat and Drosophila revealed that the N-terminal two-thirds of the sequence (especially the N-terminal approximately 20 residues) is conserved, but the C-terminal third of the sequence shows no similarity, suggesting functional and structural roles for both regions. Implications for the structural and functional aspects of MCP subunits are discussed based on the sequence similarity.     

H1 Histone and nucleosome repeat length alterations associated with the in vitro differentiation of murine embryonal carcinoma cells to extra-embryonic endoderm

The histone compositions and average distance between nucleosomes have been determined for F9.22 and PSA1 murine embryonal carcinoma cell lines, for primary extra-embryonic endoderm derived from the in vitro differentiation of PSA1 embryonal carcinoma cells, and for two long-term extra-embryonic endodermal cell lines. A change in the relative proportions of two forms of the H1 histones (H1A and H1B) was found to correlate with the extra-embryonic endodermal differentiated phenotype. The embryonal carcinoma cells had a ratio of H1A/H1B of 1.49 or greater. In contrast, extra-embryonic endoderm from either cell lines or freshly isolated from differentiating embryonal carcinoma cell cultures had a ratio of H1A/H1B of less than 0.9. Partial peptide mapping of gel purified H1A and H1B suggest the two proteins differ in primary structure. The nucleosome repeat length of the embryonal carcinoma cell lines was 196 bp of DNA. Primary extra-embryonic endoderm was found to have a value of 205 bp, but the long-term extra-embryonic endodermal cell lines had an average nucleosome repeat length of 187 bp. Since both freshly isolated primary endoderm and the long-term endodermal cell lines express differentiated functions (basement membrane glycoproteins and plasminogen activator activity), there appears to be no simple correlation between the nucleosome repeat length and the expression of these differentiated functions.     

Expression of new proteins of the intermediate filament protein family in differentiating F9 embryonal carcinoma cell cytoskeleton

Differentiation of F9 embryonal carcinoma cells by retinoic acid treatment results in extraembryonic endoderm-like cells. The effects of this process on the protein composition of the intermediate filaments were studied by two-dimensional gel electrophoresis and by immunoblotting. By this approach, two new proteins induced in differentiating cells, p57 and p54, were identified in cytoskeletal preparations enriched in intermediate filaments. The 57-kDa protein could be resolved into at least three components (pI 5.6-5.9), and the 54-kDa protein into at least two components (pI approximately 5.6). Both proteins reacted with a monoclonal antibody which recognizes an antigenic determinant common to all intermediate filaments. Based on these results, the two proteins were identified as members of the intermediate filament protein family. Partial digestion with V8 protease showed that p57 was different from vimentin, another intermediate filament protein present in these cells. p57 and p54 were also immunodetected by a polyclonal anti-keratin anti-serum, which suggests that these proteins share some homology with the keratins. These two proteins are different from the endodermal cytoskeletal protein A and B (endo A and endo B) keratins, which are known to be present in extraembryonic endoderm-like cells. They were also more abundant than endo A and endo B in differentiating F9 embryonal carcinoma cells, but almost undetectable in terminally differentiated extraembryonic endoderm-like cells, where endo A and endo B are readily detectable. This suggests that p57 and p54 have a different pattern of expression than endo A and endo B.     

Selective changes in the phosphorylation of endogenous proteins in subcellular fractions from cyclic amp-induced differentiated neuroblastoma cells

The endogenous phosphorylation of specific proteins was studied in subcellular fractions from proliferating and cAMP-induced differentiated neuroblastoma cells. Fractions containing nuclear, membrane-bound, and cytosolic proteins were incubated with [-³²P]ATP, in the presence and absence of added cyclic nucleotides. Phosphate incorporation into specific proteins was determined by slab-gel electrophoresis of sodium dodecyl sulfate-solubilized reaction products. Cytosol fractions from differentiated cells demonstrated a twofold increase in cAMP-dependent phosphorylation of a specific protein with apparent mol wt of 59,000 daltons and a comparable decrease in cAMP-independent phosphorylation of another protein (97,000). The nuclear fraction of differentiated cells showed an increase in the cAMP-independent phosphorylation of two nonhistone proteins (110,000 and 102,000). Membrane fractions from differentiated cells exhibited a differential decrease in endogenous phosphorylation of specific proteins. Selective alterations in the phosphorylation of specific proteins in various subcellular components may be important biochemical events associated with the increased levels of differentiated functions in neuroblastoma cells in culture.     

Identification of fetomodulin, a surface marker protein of fetal development, as thrombomodulin by gene cloning and functional assays

Fetomodulin (FM) was previously shown to be a surface marker protein of parietal endoderm by in vitro differentiation of F9 embryonal carcinoma cells and by immunohistochemistry of in vivo embryos. BALB/3T3 and sarcoma S180 cells of the mouse were also shown to possess a protein which was indistinguishable from FM by immunological and structural criteria. We now show by protein and DNA sequencing and by functional assays that FM is identical to thrombomodulin, an anticoagulant endothelial thrombin receptor. Partial amino acid sequences of FM from S180 cells suggested homology between FM and thrombomodulin. An FM cDNA fragment was obtained by screening an expression library, which was constructed with restricted BALB/3T3 cDNA, with polyclonal anti-FM antibody. Several longer cDNA clones were than isolated using this fragment as a probe. They elucidated a 3369-bp partial sequence which encompassed 93% of the coding sequence. The remaining structure was determined from a genomic DNA clone. The deduced FM structure proved to be identical to that of thrombomodulin of mouse lung. Affinity-purified FM of BALB/3T3 and differentiated F9 cells was as active as thrombomodulin of the lung in binding thrombin and also as an anticoagulant. Structural and functional identity of the two proteins was thus confirmed. During embryonic development, FM immunoreactivity is localized not only in vasculatures but also at sites of cell-to-cell contact, including lung bud and neural epithelium, which were not expected a priori to possess this endothelial surface protein. FM may be a multifunctional protein with unique roles in embryonic development.     

Female parthenogenetic apomixis and androsporogenesis in Douglas-fir embryonal initials in an artificial sporangium

Control of female parthenogenetic apomixis and androsporogenesis of Douglas-fir embryonal initials was studied using an experimental culture system in which changes in growth condition can mediate changes in cell identity and outcomes. This culture system constitutes an artificial sporangium in which myriad culture conditions can be simulated and should be applicable for research on a variety of gymnosperms. In this study, embryonal initials from developing seeds from two Douglas-fir trees were rescued and became reprogrammed for female parthenogenetic apomixis (fPA) and parthenogenetic androsporogenesis (mPA). Female PA was initiated by endomitosis forming a binucleate cell with a diploid egg-equivalent and an apoptotic ventral canal nucleus in an archegonial tube. Egg-equivalent nuclei formed cells (parthenotes) that were discharged into an aqueous culture medium. Parthenotes developed axial tiers atypical of early embryogenesis in seeds. Earlier in the year, androsporangial tubes were parthenogenetically differentiated and released monads, dyads, triads, and tetrads into the culture medium. Spores showed chromosomal aberrations. PA demonstrated a temporal separation in gender expression (dichogamy). Embryonal initials brought forward and by-passed the long juvenile phases normally needed for cells to develop into trees and express reproductive maturity. Expressions of fPA and mPA indicated that the specialized culture flasks served as an artificial sporangium (AS). Awareness is raised for the value of an AS for research in gymnosperm life cycles and as a teaching and research laboratory.     

Digestibility of fractionated green biomass as protein source for monogastric animals

Globally, there is an increased demand for sustainable protein sources for animal feed. Grass and forage legumes have the yield potential to become such alternatives, but the protein needs to be separated from the fibres. Red clover, white clover, lucerne and perennial ryegrass were fractionated into a green juice and a fibrous pulp in a screw-press and protein was subsequently precipitated. The nitrogen (N) and amino acid composition of the produced fractions was analysed and the digestibility of dry matter (DM) and N was evaluated using a rat digestibility trial. The aim was to determine the effect of fractionation on composition and digestibility in order to evaluate the four plants as potential protein sources for monogastrics. Protein concentrates with CP concentrations of 240 to 388 g/kg DM and fibrous pulps with CP concentrations of 111 to 216 g/kg DM were produced. The sum of all analysed amino acids was highest in the protein concentrates corresponding to a low concentration of non-protein nitrogen ranging from 4.9% to 10.4%. Only small variations were seen in the amino acid compositions of the different plants and fractions. The concentration of the essential lysine and methionine in the protein concentrate ranged from 6.27 to 6.67 g/16 g N and 1.54 to 2.09 g/16 g N for lysine and methionine, respectively. For all plants species, total tract digestibility of DM and standardised N digestibility was significantly higher in the protein concentrates (60.8% to 76.5% and 75.4% to 85.0% for DM and N, respectively) compared to pulp (21.2% to 43.4% and 52.1% to 72.5% for DM and N, respectively). Digestibility of lucerne protein concentrate (76.5% and 85.0% for DM and N, respectively) was higher than of the unprocessed plant (39.6% and 74.9% for DM and N, respectively), whereas for red and white clover no difference was found. The amino acids methionine and cysteine were limiting for pigs and broilers in all fractions regardless of plant origin, and low scores were also found for lysine. The study demonstrated great potential of using green plants as a protein source for monogastrics because of high protein content, balanced amino acid composition and high digestibility of DM and N. The effects of processing and protein precipitation were pronounced in lucerne where significantly improved digestibility was observed in the protein concentrate. The results from the study provide valuable and enhanced knowledge to the production of alternative and sustainable protein sources for monogastric feed.     

Protein kinase C and phorbol ester receptor expression related to growth and differentiation of nullipotent and pluripotent embryonal carcinoma cells

We have characterized effects of phorbol, 12-myristate 13 acetate (PMA) on growth and differentiation in a nullipotent embryonal carcinoma (EC) cell line, F9, in a pluripotent EC line, P19, and in the differentiated derivatives of these cells, In P19EC and F9EC PMA addition resulted in inhibition of growth, while in the differentiated derivates PMA was mitogenic. PMA did not induce differentiation in EC cells but potentiated the retinoic acid (RA) induced differentiation in P19EC, although, not in F9EC. Rapid morphological changes by PMA were seen in P19EC and two differentiated derivatives which represent different stages of differentiation. In F9 no rapid morphological changes were induced by PMA. Using [3H]phorbol dibutyrate as a ligand we showed that during differentiation into endoderm-like cells the number of phorbol ester receptors increases, while in epithelial-like derivatives no increase is found. In differentiated cells with an increased number of phorbol ester receptors, the cytoplasmic Ca2+- and phospholipid-dependent protein kinase (the putative receptor for phorbol esters) activity was also increased. Only in those derivatives where the number of phorbol ester receptors is increased, is the binding of epidermal growth factor (EGF) inhibited by PMA. These results suggest a relationship between levels of expression of phorbol ester receptors, cytoplasmic protein kinase C and biological effects, namely rapid morphological changes, altered growth, potentiation of RA induced differentiation, and inhibition of EGF binding.