Regulation of polypeptide chain initiation and activity of initiation factor eIF-2 in Chinese-hamster-ovary cell mutants containing temperature-sensitive aminoacyl-tRNA synthetases

The regulation of polypeptide chain initiation has been investigated in extracts from a number of well-characterized Chinese hamster ovary (CHO) cell mutants containing different temperature-sensitive aminoacyl-tRNA synthetases. These cells exhibit a large decline in the rate of initiation when cultures are shifted from the permissive temperature of 34 degrees C to the non-permissive temperature of 39.5 degrees C. During a brief incubation with [35S]Met-tRNAMetf or [35S]methionine, formation of initiation complexes on native 40S ribosomal subunits and 80S ribosomes is severely impaired in extracts from the mutant cell lines exposed to 39.5 degrees C. Wild-type cells exposed to 39.5 degrees C do not show any inhibition of protein synthesis or initiation complex formation. Inhibition of formation of 40S initiation complexes in the extracts from mutant cells, incubated at the non-permissive temperature, is shown to be independent of possible changes in mRNA binding or the rate of polypeptide chain elongation and is not due to any decrease in the total amount of initiation factor eIF-2 present. However, assays of eIF-2 X GTP X Met-tRNAMetf ternary complex formation in postribosomal supernatants from the temperature-sensitive mutants reveal a marked defect in the activity of eIF-2 after exposure of the cells to 39.5 degrees C and addition of exogenous eIF-2 to cell-free protein-synthesizing systems from cells incubated at 34 degrees C and 39.5 degrees C eliminates the difference in activity between them. The activity of the initiation factor itself is not directly temperature-sensitive in the mutant CHO cells. The results suggest that the activity of aminoacyl-tRNA synthetases can affect the ability of eIF-2 to bind Met-tRNAMetf and form 40S initiation complexes in intact cells, indicating a regulatory link between polypeptide chain elongation and chain initiation.     

Initiation of mammalian protein synthesis: dynamic properties of the assembly process in vitro

Binding of the Met-tRNAMetf . eIf-2 GTP complex to the 40 S ribosomal subunit is the first step in initiation of eukaryotic protein synthesis. The extent of binding and the stability of the complex are enhanced by initiation factors eIF-3 and eIF-4C, AUG and elevated magnesium concentration. The reversibility of reaction steps occurring during the assembly of the initiation complex is measured as the rate of Met-tRNAMetf exchange in the initiation complex and its intermediates. This rate progressively decreases and Met-tRNAMetf binding becomes irreversible upon binding of mRNA. The association of the 40 S Met-tRNAMetf mRNA initiation complex with the 60 S ribosomal subunit is again reversible as long as elongation does not occur.     

Immunologically distinct binding molecules for progesterone and RU38486 in the chick oviduct cytosol

[3H]Progesterone and [3H]RU38486 binding in the chick oviduct cytosol is associated with macromolecules which sediment as 8 S and 4 S moieties, respectively, in molybdate-containing 5-20% sucrose gradients. The [3H]progesterone binding could be displaced by excess progesterone, but not by RU38486. Conversely, the [3H]RU38486 binding was able to compete with RU38486 but not by excess progesterone. A preparation containing antibodies against chick oviduct progesterone receptor recognized only the [3H]progesterone-receptor complex but not the 4 S, [3H]RU38486 binding component of the chick cytosol. In the calf uterus cytosol, [3H]R5020 (a synthetic progestin) and [3H]RU38486 were associated with 8 S molecules and the peaks of radioactivity were displaceable upon preincubation with radionert steroids. In addition, the complexes were recognized by antibodies to chick oviduct progesterone receptor. Our data suggest that in the chick oviduct cytosol, RU38486 does not bind to progesterone receptor, but interacts with an immunologically distinct macromolecule.     

Binding of protein synthesis initiation factor IF1 to 30 S ribosomal subunits: effects of other initiation factors and identification of proteins near the binding site.

The effects of other components of the initiation complex on Escherichia coli initiation factor IFI binding to 30 S ribosomal subunits were studied. Binding of [¹⁴C]IF1 in the absence of other initiation complex components was slight. Addition of either IF2 or IF3 stimulated binding to a variable extent. Maximum binding was observed when both IF2 and IF3 were present. Addition of GTP, fMet-tRNA, and phage R17 RNA caused little or no further stimulation of [¹⁴C]IF1 binding. A maximum of 0.5 molecule of [¹⁴C]IF1 bound per 30 S subunit in the presence of an excess of each of the three factors over 30 S subunits.Complexes of 30 S subunits, [¹⁴C]IF1, IF2, and IF3 were treated with the bifunctional protein cross-linking reagent dimethyl suberimidate in order to identify the ribosomal proteins near the binding site for IF1. Non-cross-linked [¹⁴C]IF1 was removed from the complexes by sedimentation through buffer containing a high salt concentration, and total protein was extracted from the pelleted particles. Approximately 12% of the [¹⁴C]IF1 was recovered in the pellet fraction. The mixture of cross-linked products was analyzed by polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Autoradiography of the gel showed radioactive bands with molecular weights of 21,000, 25,000, and many greater than 120,000. The results indicate that [¹⁴C]IF1 was cross-linked directly to at least two ribosomal proteins. Analysis of the cross-linked mixture by radioimmunodiffusion with specific antisera prepared against each of the 30 S ribosomal proteins showed radioactivity in the precipitin bands formed with antisera against S12 and S19, and in lower yield with those against S1 and S13. Antiserum against IF2 also showed [¹⁴C]IF1 in the precipitin band. The results show that [¹⁴C]IF1 was present in covalently cross-linked complexes containing 30 S ribosomal proteins S1, S12, S13 and S19, and initiation factor IF2. The same ribosomal proteins have been implicated in the binding sites for IF2 and IF3. The results suggest that the three initiation factors bind to the 30 S subunit at the same or overlapping sites.     

Identification of proteins of the 40 S ribosomal subunit involved in interaction with initiation factor eIF-2 in the quaternary initiation complex by means of monospecific antibodies

Monospecific polyclonal antibodies against seven proteins of the 40 S subunit of rat liver ribosomes were used to identify ribosomal proteins involved in interaction with initiation factor eIF-2 in the quaternary initiation complex [eIF-2 X GMPPCP X [3H]Met-tRNAf X 40 S ribosomal subunit]. Dimeric immune complexes of 40 S subunits mediated by antibodies against ribosomal proteins S3a, S13/16, S19 and S24 were found to be unable to bind the ternary initiation complex [eIF-2 X GMPPCP X [3H]Met-tRNAf]. In contrast, 40 S dimers mediated by antibodies against proteins S2, S3 and S17 were found to bind the ternary complex. Therefore, from the ribosomal proteins tested, only proteins S3a, S13/16, S19 and S24 are concluded to be involved in eIF-2 binding to the 40 S subunit.     

GTP hydrolysis during methionyl-tRNAf binding to 40 S ribosomal subunits and the site of edeine inhibition.

Three lines of evidence are presented indicating that GTP hydrolysis associated with eukaryotic peptide initiation occurs in the absence of 60 S subunits when methionyl-tRNAf is bound to 40 S ribosomal subunits. An enzyme fraction required for binding of methionyl-tRNAf to 40 S subunits and peptide initiation, tentatively equated with eIF-(4 + 5), has GTPase activity and appears to be responsible for hydrolysis of GTP in the methionyl-tRNAf.eIF-2.GTP complex. Direct analysis of the methionyl-tRNAf.40 S complex formed with with eIF-2 and [8-3H] guanine, [gamma-32P]GTP reveals bound guanine but not gamma-phosphate. Edeine, a peptide antibiotic containing spermidine and beta-tyrosine residues at its COOH terminus and NH2 terminus, respectively, blocks peptide initiation and interferes with binding of methionyl-tRNAf to 40 S ribosomal subunits. Inhibition of binding is observed when the eIF-2-mediated binding reaction is carried out with GTP but not with guanosine 5'-(beta,gamma-methylene)triphosphate or guanosine 5'-(beta,gamma-imido)triphosphate. Edeine was labeled by iodination and shown to bind with high affinity to 40 S but not to 60 S ribosomal subunits. It is suggested that edeine blocks a specific site on the 40 S ribosomal subunit to which a segment of the methionyl-tRNAf molecule is bound during the course of the initiation reaction sequence.     

Intestinal cytosol binders of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3

The binding of 25-hydroxy-[26,27-³H]vitamin D₃ and 1,25-dihydroxy-[26,27-³H]vitamin D₃ to the cytosol of intestinal mucosa of chicks and rats has been studied by sucrose gradient analysis. The cytosol from chick mucosa showed variable binding of 1,25-dihydroxyvitamin D₃ to a 3.0S macromolecule which has high affinity and low capacity for this metabolite. However, when the mucosa was washed extensively before homogenization, a 3.7S macromolecule was consistently observed which showed considerable specificity and affinity for 1,25-dihydroxyvitamin D₃. Although 3.7S binders for 1,25-dihydroxyvitamin D₃ could also be located in other organs, competition experiments with excess nonradioactive 1,25-dihydroxyvitamin D₃ suggested that they were not identical to the 3.7S macromolecule from intestinal mucosal cytosol. As the 3.7S macromolecule was allowed to stand at 4 °C with bound 1,25-dihydroxy-[³H]vitamin D₃, the 1,25-dihydroxy-[³H]vitamin D₃ became increasingly resistant to displacement by non-radioactive 1,25-dihydroxyvitamin D₃. The 1,25-dihydroxy-[³H]vitamin D₃ remained unchanged and easily extractable with lipid solvents through this change, making unlikely the establishment of a covalent bond. Unlike the chick, mucosa from rats yielded cytosol in which no specific binding of 1,25-dihydroxy-[³H]vitamin D₃ was detected. Instead, a 5-6S macromolecule which binds both 1,25-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃ was found. This protein which was also found in chick mucosa shows preferential binding for 25-hydroxyvitamin D₃. It could be removed by washing the mucosa with buffer prior to homogenization which suggests that it may not be a cytosolic protein. Although the 3.7S protein from chick mucosa has properties consistent with its possible role as a receptor, the 5-6S macromolecule does not appear to have “receptor”-like properties.     

Cross-linking of initiation factor IF3 to proteins of the Escherichia coli 30 S ribosomal subunit

Complexes of 30 S subunits and [¹⁴C]IF3 were allowed to react with the protein cross-linking reagents, N,N′-p-phenylenedimaleimide or dimethylsuberimidate. Non-cross-linked IF3 was removed from the complex by centrifugation in a buffer containing a high salt concentration, and the total protein was extracted from the pelleted particles. The mixture of cross-linked products was analyzed by radioimmunodiffusion with antisera prepared against all of the individual 30 S ribosomal proteins. Radioactivity was found in the precipitin bands formed with antisera against ribosomal proteins S1, S11, S12, S13, S19 and S21. The results show that IF3 was present in covalent cross-linked complexes containing those 30 S ribosomal proteins and imply that they comprise or are near the binding site for initiation factor IF3.     

Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors

The assembly of initiation complexes is studied in a protein synthesis initiation assay containing ribosomal subunits, globin [¹²⁵I]mRNA, [³H]Met-tRNA_f, seven purified initiation factors, ATP and GTP. By omitting single components from the initiation assay, specific roles of the initiation factors, ATP and GTP are demonstrated. The initiation factor eIF-2 is required for the binding of Met-tRNA_f to the 40 S ribosomal subunit. The initial Met-tRNA_f binding to the small ribosomal subunit is a stringent prerequisite for the subsequent mRNA binding. The initiation factors eIF-3, eIF-4A, eIF-4B and eIF-4C together with ATP promote the binding of mRNA to the 40 S initiation complex. The association of the 40 S initiation complex with the 60 S ribosome subunit to form an 80 S initiation complex is mediated by the initiation factor eIF-5 and requires the hydrolysis of GTP. The factor eIF-1 gives a twofold overall stimulation of initiation complex formation. A model of the sequential steps in the assembly of the 80 S initiation complex in mammalian protein synthesis is presented.     

Electron microscopic study of eukaryotic 40S initiation complex in protein synthesis

This electron microscopic study demonstrates that formation of a functional eukaryotic 40S initiation complex is accompanied by conformational changes which obscure the characteristic structural features of the 40S ribosomal subunits and of the initiation factor eIF-3, the only macromolecular components of the complex individually resolvable by conventional high resolution electron microscopy. The complex, characterized by a sedimentation coefficient of 46S, appears as a globular particle with a diameter of about 280 A and several characteristic protrusions and incisions. Similar structures were obtained with [40S X eIF-3] initiation complexes formed by interaction of eIF-3 from rabbit reticulocytes with 40S ribosomal subunits from either A. salina cysts or mouse liver. Incubation of eIF-3 with prokaryotic 30S subunits from E. coli produced no [30S X eIF-3] structures. The binding of eIF-3 to 40S subunits is weak, and both the [40S X eIF-3] and the complete 40S initiation complexes have to be stabilized by glutaraldehyde fixation. The extensive conformational changes associated with the complex formation preclude direct electron microscopic localization of eIF-3, a globular protein approximately 100 A in diameter, in the initiation domain of the 40S subunit.     

Evidence for a 1,25-dihydroxycholecalciferol-dependent spermine-binding protein in chick duodenal mucosa

The spermine-binding activity of a cytosol protein fraction from chick duodenal mucosa changes in relation to the circulating level of 1,25-dihydroxycholecalciferol. The spermine-binding activity increases very rapidly within 1–2 hours after the rachitic chick was dosed intracardially with 1,25-dihydroxycholecalciferol. The clear and reproducible response is prevented by actinomycin D and cycloheximide. This increase is one of the earliest events induced by the active form of vitamin D₃ in the duodenal cell of rachitic chicks.     

The 60 S ribosomal subunit as a carrier of eukaryotic initiation factor 2 and the site of reversing factor activity during protein synthesis

Studies on the recycling of eukaryotic initiation factor 2 (eIF-2) during protein synthesis in normal and heme-deficient reticulocyte lysates indicate that eIF-2 binds physiologically to the 60 S ribosomal subunit. Several findings suggest that the 60 S subunit serves as a carrier for eIF-2 during protein synthesis. The addition of purified eIF-2 (beta-32P) to normal hemin-supplemented lysates results in its binding to polyribosomal 60 S subunits; the binding is temperature-dependent. In lysates inhibited by heme deficiency, phosphorylated eIF-2 alpha can be detected on polyribosomal 60 S subunits early in the initial linear phase of protein synthesis; after polyribosomal disaggregation and shut-off of protein synthesis, phosphorylated eIF-2 alpha accumulates on free 60 S ribosome subunits and on the 60 S subunits of 80 S ribosome couples. The phosphorylated eIF-2 alpha associated with the 60 S subunits in heme-deficient lysates appears to be present as the binary complex [eIF-2 (alpha P) X GDP]; the binding of this complex to the 60 S subunit is tight and is not affected by treatment with 25 mM EDTA or by sedimentation in sucrose gradients. Reversal of the inhibition of protein synthesis in heme-deficient lysates by the addition of reversing factor results in a rapid binding of reversing factor to the 60 S subunits and a concomitant dissociation of [eIF-2(alpha P) X GDP]. These findings suggest that the [eIF-2 X GDP] binary complex formed during the assembly of the 80 S initiation complex binds to the 60 S subunit of polyribosomes and is subsequently released by the action of reversing factor.     

Preparation and characterization of a cell-free system from Chinese hamster ovary cells that translates natural messenger ribonucleic acid and analysis of intermediary reactions

A cell-free system from cultured Chinese hamster ovary cells has been developed, which translates endogenous mRNAs, exogenous natural mRNAs, and synthetic polynucleotide templates. The analysis of most of the reactions involved in initiation, elongation, and termination of protein synthesis can be carried out in this system. The postmitochondrial fraction, containing ribosomal 40 and 60 S subunits, 80 S ribosomes, polysomes, and cytosol proteins, incorporates amino acids into protein. The preparation is capable of recycling endogenous mRNA by initiating protein synthesis on polysomal mRNA, and of initiating protein synthesis on exogenous templates. When endogenous mRNA is degraded with micrococcal nuclease, polysomes are no longer evident and protein synthesis is markedly depended on added mRNA, ATP, GTP, and a nucleoside triphosphate-generating system. Amino acid incorporation is linear for over 2 h, polysomes containing nascent polypeptide chains are reformed and, with time, most of the protein synthesized is released into the media. Gel electrophoretic analysis of the product formed in response to globin mRNA indicates that most of the radioactivity migrates as a single peak, in the region corresponding to globin. Comparison of the electrophoretic pattern obtained from labeled Chinese hamster ovary cells with that from incubations of cell extract and Chinese hamster ovary mRNA indicates that essentially all of the polypeptides formed by the intact cell are synthesized by the cell-free system. Sucrose gradient centrifugation of incubations containing mRNA-depleted extract and [³⁵S]methionine, in the absence of added mRNA, is used to detect initiation intermediates in the formation of the [40 S Met-tRNA_f] complex and, with added natural mRNA plus cycloheximide, to detect intermediates in the formation of the 80 S initiation complex. Chain elongation reactions are measured by the incorporation of [³H]phenylalanine into polyphenylalanine in extracts supplemented with poly(U), or by the formation of nascent polypeptide chains on polysomes with natural mRNA. Chain termination is measured by analyzing the amount of radioactive protein released into the cytosol.     

Binding of chloramphenicol and a fragment of aminoacyl-transfer ribonucleic acid to ribosomes and a ribosome precursor from a mutant of Escherichia coli.

During exponential growth, the mutatn strain Escherichia coli 15-28 accumulates 47S particles, which are unusual precursors to 50S ribosomal subunits. The 47S particles have little ability to bind chloramphenicol, but binding of a fragment of aminoacyl-tRNA is about half that by completed subunits. The 70S (and 50S) ribosomes of strain 15-28 and its parent (strain 15TP) do not differ in chloramphenicol binding. Although ribosomes from the mutant are less able than those from the parent to bind the fragment, this difference is not as marked as was found previously [Sims & Wild (1976) Biochem. J. 160, 721-726] for the binding of an analogue of peptidyl-tRNA and for peptidyltransferase activity. The altered activities may arise because strain 15-28 misassembles 50S subunits of altered conformation and because the few proteins that 47S patricles lack have vital functions in some of the partial reactions of protein synthesis.     

Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation.

The AUG-dependent formation of an 80 S ribosomal initiation complex was studied using purified rabbit reticulocyte initiation factors radiolabeled by reductive methylation. The radiolabeled initiation factors were as biologically active as untreated factors. Reaction mixtures containing a variety of components (AUG, GTP, Met-tRNAf, initiation factors, and 40 S and 60 S ribosomal subunits) were incubated at 30 degrees C and then analyzed on linear sucrose gradients for the formation of ribosomal complexes. The results show that both eukaryotic initiation factor (eIF)-3 and the ternary complex (eIF-2.GTP.Met-tRNAf) bind independently to the 40 S subunit and each of these components enhances the binding of the other. All of the polypeptides of eIF-2 and eIF-3 participate in this binding. Formation of an 80 S ribosomal complex requires eIF-5 and 60 S subunits in a reaction that is stimulated by eIF-4C. Both eIF-2 and eIF-3 are released from the 40 S preinitiation complex during formation of the 80 S initiation complex. Release of eIF-2 and eIF-3 does not occur and 80 S ribosomal complexes are not formed if GTP is replaced by a nonhydrolyzable analog such as guanosine 5'-O3-(1,2-mu-imido)triphosphate. Despite a variety of attempts, it has not yet been possible to demonstrate binding of eIF-4C, eIF-4D, or eIF-5 to either 40 S or 80 S ribosomal complexes.     

The protein translocation channel binds proteasomes to the endoplasmic reticulum membrane

Misfolded secretory proteins are transported across the endoplasmic reticulum (ER) membrane into the cytosol for degradation by proteasomes. A large fraction of proteasomes in a cell is associated with the ER membrane. We show here that binding of proteasomes to ER membranes is salt sensitive, ATP dependent, and mediated by the 19S regulatory particle. The base of the 19S particle, which contains six AAA-ATPases, binds to microsomal membranes with high affinity, whereas the 19S lid complex binds weakly. We demonstrate that ribosomes and proteasomes compete for binding to the ER membrane and have similar affinities for their receptor. Ribosomes bind to the protein conducting channel formed by the Sec61 complex in the ER membrane. We co-precipitated subunits of the Sec61 complex with ER-associated proteasome 19S particles, and found that proteoliposomes containing only the Sec61 complex retained proteasome binding activity. Collectively, our data suggest that the Sec61 channel is a principal proteasome receptor in the ER membrane.     

Identification of the ribosomal proteins present in the vicinity of globin mRNA in the 40S initiation complex

The interaction of ribosomal proteins with mRNA in the 40S initiation complex was examined by chemical cross-linking. 40S initiation complexes were formed by incubating rat liver [(3)H]Met-tRNAi, rat liver 40S ribosomal subunits, rabbit globin mRNA, and partially purified initiation factors of rabbit reticulocytes in the presence of guanylyl(beta, gamma-methylene)-diphosphonate. The initiation complexes were then treated with 1,3-butadiene diepoxide to introduce crosslinks between the mRNA and proteins. The covalent mRNA-protein conjugates were isolated by chromatography on an oligo(dT) cellulose column in the presence of sodium dodecyl sulfate, followed by sucrose density gradient centrifugation. Proteins cross-linked to the mRNA were labeled with Na(125)I, extracted by extensive ribonuclease digestion, and analyzed by two-dimensional and diagonal polyacrylamide gel electrophoresis. Three ribosomal proteins, S6, S8, and S23/S24, together with small amounts of S3/S3a, S27, and S30, were identified as the protein components cross-linked to the globin mRNA protein complex, and were shown to attach directly to the mRNA. It is suggested that these proteins constitute the ribosomal binding site for mRNA in the 40S initiation complex.     

Iodohexestrols. II. Characterization of the binding and estrogenic activity of iodinated hexestrol derivatives, in vitro and in vivo.

The affinity of ortho-iodinated hexestrols for the estrogen binding protein from rat uterus, determined by competitive binding assay, decreases with progressive iodine substitution; 3-iodohexestrol (I-Hex) has a binding affinity 42% that of estradiol. Analysis of [3-H]-I-Hex binding in rat uterine cytosol by sucrose density gradient centrifugation shows both an estrogen-specific binding component (8 S) and a more abundant component (4 S) that is not estrogen specific. Scatchard analysis indicates that this latter binding is of high affinity (Kd equals to 3.7-8.3 times 10- minus-9 M) but is not uterine specific. Polyacrylamide gel electrophoresis shows that most of the [3-H]-I-Hex binding activity in serum and uterine cytosol is distinct from and anodic to the principal protein component (albumin), and that is comigrates with [14-C]thyroxine binding activity. In in vitro incubation of rat uteri, I-Hex can block the specific uptake of [3-H]estradiol into the nuclear fraction; it itself causes a translocation of estrogen-specific binding capacity (as measured by exchange) from cytoplasm to nuclei, and can induce the synthesis of an estrogen-specific uterine protein, all under conditions where it is not metabolically deiodinated to hexestrol. The uterotrophic activities of the iodohexestrols are in most cases comparable to that expected on the basis of their competitive binding affinities. However, selective, estrogen-specific uptake of [3-H]-I-Hex into rat uterus, either in vitro or in vivo, cannot be demonstrated.     

Final Pre-40S Maturation Depends on the Functional Integrity of the 60S Subunit Ribosomal Protein L3

Ribosomal protein L3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles. We report that the rpl3[W255C] allele, which affects the affinity and function of translation elongation factors, impairs cytoplasmic maturation of 20S pre-rRNA. This was not seen for other mutations in or depletion of L3 or other 60S ribosomal proteins. Surprisingly, pre-40S particles containing 20S pre-rRNA form translation-competent 80S ribosomes, and translation inhibition partially suppresses 20S pre-rRNA accumulation. The GTP-dependent translation initiation factor Fun12 (yeast eIF5B) shows similar in vivo binding to ribosomal particles from wild-type and rpl3[W255C] cells. However, the GTPase activity of eIF5B failed to stimulate processing of 20S pre-rRNA when assayed with ribosomal particles purified from rpl3[W255C] cells. We conclude that L3 plays an important role in the function of eIF5B in stimulating 3′ end processing of 18S rRNA in the context of 80S ribosomes that have not yet engaged in translation. These findings indicate that the correct conformation of the GTPase activation region is assessed in a quality control step during maturation of cytoplasmic pre-ribosomal particles.     

Selective retention and formation of a delta5-androstenediol-receptor complex in cell nuclei of the rat vagina.

Cellular protein binding of a number of androstene and androstane derivatives that promote the growth of the vagina in rats has been studied. It was found that cell nuclei of the rat vagina contain a tissue-specific protein that binds 3beta,17beta-dihydroxy-androst-5-ene (delta5-androstenediol), a unique steroid causing growth and keratinization of the vaginal epithelium. The formation of the steroid-protein complex can be demonstrated by the administration of delta5-[3H]androstenediol to ovariectomized rats or by the incubation of minced vagina with the radioactive steroid. The steroid can interact with purified vaginal cell nuclei even in the absence of a cytosol preparation, forming the same steroid-protein complex. The formation of the complex is temperature-dependent; it occurs much more readily at 37 degrees than at 0 degrees. The delta5-[3H]androstenediol-protein complex migrated as about 4 S in a sucrose gradient medium containing 0.4 M KCl. A similar complex can be detected when nuclei of vaginal cells are incubated with 3alpha,17beta-dihydroxy-5alpha-androstane, 3beta,17beta-dihydroxy-5alpha-androstane, and 3beta-hydroxy-androst-5-en-17-one which also have the capability of stimulating vaginal epithelium, although in somewhat different ways. These steroids may bind to different groups of chromatin-bound receptor proteins in various layers of vaginal epithelium. The delta5-androstenediol binding protein is not found in the vaginal cytosol fraction that contains receptor proteins for estrogens and progestins, nor in the cytosol or nuclei of rat uterus cells, but not in muscle, brain, kidney, or liver. Testosterone and 5alpha-dihydrostestosterone bind weakly to the protein, whereas cortisol, androstenedione, 17beta-estradiol, and progesterone do not bind to the same protein by any significant extent.