Molecular cloning and characterization of rat estrogen receptor cDNA.

A cDNA clone of rat uterus estrogen receptor (ER) has been isolated and sequenced. This clone contains a complete open reading frame encoding 600 amino acid residues which is 5 and 11 amino acids larger than the corresponding molecules of human and chicken, respectively. The molecular weight of this protein is calculated to be 67,029. When this clone was ligated to the pSV2 vector and transfected into COS7 cells, a protein was produced that had the same affinity to estrogen as rat uterus ER. This sequence shows 88% homology with human ER; 528 amino acids are identical and 14 amino acids are conservative substitutions. The comparison of rat, human and chicken ER sequences indicate the presence of three highly conserved regions suggesting that these regions play important roles in ER function. The putative DNA-binding domain is completely identical in rat, human and chicken. The C-terminal half region which is thought to be the estrogen binding domain is also highly conserved and is rich in hydrophobic amino acid residues. Southern blot analysis of genomic DNA with ER cDNA as a probe has shown that related sequences are present in the genome.     

Nuclear localization of the PEP protein tyrosine phosphatase.

PEP is an intracellular protein tyrosine phosphatase expressed primarily by cells of hematopoietic origin that can be divided structurally into a catalytic domain and a large carboxy-terminal domain. The carboxy-terminal domain is enriched in proline, glutamic acid, serine, and threonine residues (PEST sequences) and contains a nonperfect tandem repeat sequence enriched in proline residues and a carboxy terminus enriched in basic amino acids. Here we show that PEP is diffusely expressed in lymphoid tissues, consistent with expression by many different cell types. Analysis of the PEP protein identifies a nuclear localization sequence within the extreme carboxy terminus. Transfer of 18 amino acids from the carboxy terminus of PEP to beta-galactosidase conferred nuclear localization, indicating that this sequence was sufficient for nuclear localization. Proteins enriched in PEST sequences are often rapidly degraded. However, pulse-chase analysis indicates that PEP has a half-life of greater than 5 h.     

Structure and developmental expression of the nerve growth factor receptor in the chicken central nervous system

Chicken nerve growth factor (NGF) receptor cDNAs have been isolated and sequenced in an effort to identify functionally important receptor domains and as an initial step in determining the functions of the NGF receptor in early embryogenesis. Comparisons of the primary amino acid sequences of the avian and mammalian NGF receptors have identified several discrete domains that differ in their degree of conservation. The highly conserved regions include an extracellular domain, likely to be involved in ligand binding, in which the positions of 24 cysteine residues and virtually all negatively charged residues are conserved; a transmembrane region, including flanking stretches of extracellular and cytoplasmic amino acids, which has properties suggesting it interacts with other proteins; and a cytoplasmic PEST sequence, which may regulate receptor turnover. Transient expression of NGF receptor mRNA has been seen in many regions of the developing CNS. Experiments suggest that both NGF and its receptor help regulate development of the retina.     

Computer-based search for steroid and DNA binding sites on estrogen and glucocorticoid receptors

The primary amino acid sequences of proteins that are receptors for estrogen, glucocorticoids, and ouabain were compared with each other using computer programs designed to detect and quantify similarities between proteins. Three regions of similarity between the estrogen receptor (ER) and the glucocorticoid receptor (GR) were identified. On the ER, residues 173-250, 323-395, and 426-458 are similar to residues 409-486, 540-612, and 644-676, respectively, on the GR. The ALIGN computer analysis of these segments on the ER and the GR gave comparison scores that were 16.8, 13.7, and 6.8 standard deviations higher, respectively, than that obtained with a comparison of randomized sequences of these proteins. The probability of getting these scores by chance is less than 10(-60), 10(-40), and 10(-11), respectively. Others have proposed that the segment on the ER and GR that is nearest their amino terminus (e.g. residues 173-250 of the ER) is part of their DNA binding domain and that the other two similar segments on each receptor, which are closer to their carboxy terminus, are part of their steroid binding domain. Here, we present evidence to support both of these hypotheses. First, an Align computer analysis indicates that residues 323-395 of the ER and residues 570-612 of the GR contain a region that is similar to a part of the alpha-subunit of the (Na+ + K+)ATPase that is hypothesized to bind the steroid ouabain. This similarity provides additional support for the proposed location of the steroid binding site on the ER, GR, and (Na+ + K+)ATPase. Second, a computer search of the protein sequence database revealed that protamine, a DNA binding protein, has some similarity to residues 255-281 of the ER, which are thought to be part of the DNA binding domain in the ER. Further, we find that residues 276-281 of the ER contain a structure that has been found at the nucleotide binding domain of some protein kinases. If this region on the ER binds ATP, then it may be involved in phosphorylation/dephosphorylation of the ER, which is thought to be important in its mechanism of action.     

Structural requirements for high affinity ligand binding by estrogen receptors: a comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells.

In order to better understand the structural requirements for effective high affinity binding of estrogens and antiestrogens by the human estrogen receptor (ER), a comparative study was undertaken in which we examined: 1) native ER from the MCF-7 ER-positive human breast cancer cell line; 2) full length ER expressed in yeast; 3) the ER hormone binding domain (amino acid residues 302-595) expressed in yeast; 4) a bacterially expressed protein A fusion product encoding a truncated ER (amino acid residues 240-595); and 5) a synthetic peptide encompassing amino acids 510-551 of the ER. The binding parameters studied included affinity, kinetics, structural specificity for ligands, and stability. Full length ER expressed in yeast was very similar to the MCF-7 ER in its affinity [dissociation constant (Kd), 0.35 +/- 0.05 nM], dissociation rate (t1/2, 3-4 h at 25 C), and structural specificity for both reversible and covalently attaching affinity ligands. While the truncated ER expressed in yeast was similar to MCF-7 ER in its specificity of ligand binding, it showed a slightly reduced affinity for estradiol (Kd, 1.00 +/- 0.17 nM). The bacterially expressed ER also had a lower affinity for estradiol (Kd, 1.49 +/- 0.16 nM), which may be due in part to an increase in the dissociation rate (t1/2, 0.5 h at 25 C). The attachment of covalent affinity ligands and structural specificity for a variety of reversible ligands was comparable in the bacterially expressed ER to that observed for the receptors expressed in MCF-7 cells and yeast.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the carboxyl-terminal domain of the rat glucose-dependent insulinotropic polypeptide (GIP) receptor. A role for serines 426 and 427 in regulating the rate of internalization.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone involved in the regulation of insulin secretion. In non-insulin-dependent diabetes mellitus insulin responses to GIP are blunted, possibly due to altered signal transduction or reduced receptor number. Site-directed mutagenesis was used to construct truncated GIP receptors to study the importance of the carboxyl-terminal tail (CT) in binding, signaling, and receptor internalization. Receptors truncated at amino acids 425, 418, and 405, expressed in COS-7 or CHO-K1 cells, exhibited similar binding to wild type receptors. GIP-dependent cAMP production with the 405 mutant was decreased in COS-7 cells. Maximal cAMP production in CHO-K1 cells was reduced with all truncated forms. Binding was undetectable with a receptor truncated at amino acid 400; increasing tail length by adding 5 alanines restored binding and signaling. Mutants produced by alanine scanning of residues 394-401, adjacent to transmembrane domain 7, were all functional. CT truncation by 30 or more amino acids, mutation of serines 426/427, singly or combined, or complete CT serine knockout all reduced receptor internalization rate. The majority of the GIP receptor CT is therefore not required for signaling, a minimum chain length of approximately 405 amino acids is needed for receptor expression, and serines 426 and 427 are important for regulating rate of receptor internalization.     

Endocytosis of the transferrin receptor requires the cytoplasmic domain but not its phosphorylation site

The transferrin receptor (TR) mediates cellular iron uptake by bringing about the endocytosis of transferrin. We investigated whether the cytoplasmic domain of 65 N-terminal amino acids or phosphorylated sites within this domain constitute a structure that is required for TR endocytosis. To test this hypothesis, we modified the cytoplasmic serine residues or introduced a deletion of 36 amino acids by in vitro mutagenesis of a cDNA expression vector for human TR. Upon expression in transfected mouse Ltk- cells, both the wild-type and phosphorylation site mutant receptors mediated transferrin internalization, whereas the truncated receptor did not. These results provide evidence that the cytoplasmic domain, or part of it, is essential for internalization of the TR, but argue against a role for receptor phosphorylation in endocytosis.     

Chemical characterization by protein sequence analysis of the bovine estrogen receptor

Tryptic peptides generated from bovine estrogen receptor have been fractionated and purified using microbore column high performance liquid chromatography. Sequence analysis performed on six of these peptides, derived from diverse structural regions of the receptor protein, yielded 73 unique assignments corresponding to approximately 12% of the molecule. The amino acid sequences of these peptides displayed a high degree of similarity with corresponding sequences from estrogen receptors of mammalian origin, but were only moderately conserved in receptors from non-mammalian species. The sequenced residues of one tryptic peptide, positioned in the estrogen binding domain, were fully conserved in all estrogen receptors.     

DNA binding analysis of glucocorticoid receptor specificity mutants.

The glucocorticoid receptor (GR) DNA binding domain consists of several conserved amino acids and folds into two zinc finger-like structures. Previous transactivation experiments indicated that three amino acids residing in this region, Gly, Ser and Val, appear to be critical for target-site discrimination. Based on the solved crystal structure, these residues are at the beginning of an amphipathic alpha-helix that interacts with the DNA's major groove; of these, only valine, however, contacts DNA. In order to examine their functional role directly, we have substituted these residues for the corresponding amino acids from the estrogen receptor (ER), overexpressed and purified the mutant proteins, and assayed their binding specificity and affinity by gel mobility shifts using glucocorticoid or estrogen response elements (GRE or ERE, respectively) as DNA probes. We find that all three residues are indeed required to fully switch GR's specificity to an ERE. The contacting valine in GR is of primary importance. The corresponding residue in ER, alanine, is less important for specificity, while glutamic acid, four amino acids towards the N-terminus, is most critical for ER discrimination. Finally, we show that the GR DNA binding domain carrying all three ER-specific mutations has a significantly higher affinity for an ERE than the ER DNA binding domain itself. We interpret these results in the context of both the data presented here and the crystal structure of the GR DNA binding domain complexed to a GRE.     

The interaction site for tamoxifen aziridine with the bovine estrogen receptor

Calf uterine estrogen receptor was covalently labeled with [3H]tamoxifen aziridine during affinity chromatography purification. After carboxymethylation, affinity labeled receptor was digested with trypsin under limit conditions and the labeled peptides were fractionated by reversed-phase high performance liquid chromatography into one major and two minor components. Sequence analysis of the dominant labeled fragment indicated the facile cleavage of label during Edman degradation but identified two peptides, both derived from the extreme carboxyl terminus of the steroid-binding domain. The 17 residues of one peptide were fully conserved in all estrogen receptors. This fragment contained five nucleophilic amino acids and was considered as the more favored interaction site for tamoxifen aziridine. A corresponding region of the glucocorticoid receptor has recently been identified as one of three major contact sites for glucocorticoids (Carlstedt-Duke, J., Str?mstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J.-A., and J?rnvall, H. (1988) J. Biol. Chem. 263, 6842-6846). A comparison of amino acid physical characteristics in the hormone-binding domains of human estrogen and glucocorticoid receptors demonstrated an excellent structural correlation between the two regions and delineated elements in the estrogen receptor which may be directly involved in estradiol binding.     

Sequence requirements for trafficking of the CRAM transmembrane protein to the flagellar pocket of African trypanosomes

CRAM is a cysteine-rich acidic transmembrane protein, highly expressed in the procyclic form of Trypanosoma brucei. Cell surface expression of CRAM is restricted to the flagellar pocket of trypanosomes, the only place where receptor mediated endocytosis takes place in the parasite. CRAM can function as a receptor and was hypothesized to be a lipoprotein receptor of trypanosomes. We study mechanisms involved in the presentation and routing of CRAM to the flagellar pocket of insect- and bloodstream-form trypanosomes. By deletional mutagenesis, we found that deleting up to four amino acids from the C terminus of CRAM did not affect the localization of CRAM at the flagellar pocket. Shortening the CRAM protein by 8 and 19 amino acids from the C terminus resulted in the distribution of the CRAM protein in the endoplasmic reticulum (ER) (the CRAM protein is no longer uniquely sequestered at the flagellar pocket). This result indicates that the truncation of the CRAM C terminus affected the transport efficiency of CRAM from the ER to the flagellar pocket. However, when CRAM was truncated between 29 and 40 amino acids from the C terminus, CRAM was not only distributed in the ER but also located to the flagellar pocket and spread to the cell surface and the flagellum. Replacing the CRAM transmembrane domain with the invariant surface glycoprotein 65-derived transmembrane region did not affect the flagellar pocket location of CRAM. These results indicate that the CRAM cytoplasmic extension may exhibit two functional domains: one domain near the C terminus is important for efficient export of CRAM from the ER, while the second domain is of importance for confining CRAM to the flagellar pocket membrane.     

The COOH-terminal domain of wild-type Cot regulates its stability and kinase specific activity

Cot, initially identified as an oncogene in a truncated form, is a mitogen-activated protein kinase kinase kinase implicated in cellular activation and proliferation. Here, we show that this truncation of Cot results in a 10-fold increase in its overall kinase activity through two different mechanisms. Truncated Cot protein exhibits a lower turnover rate (half-life, 95 min) than wild-type Cot (half-life, 35 min). The degradation of wild-type and truncated Cot can be specifically inhibited by proteasome inhibitors in situ. The 20S proteasome also degrades wild-type Cot more efficiently than the truncated protein. Furthermore, the amino acid 435 to 457 region within the wild-type Cot COOH-terminal domain confers instability when transferred to the yellow fluorescent protein and targets this fusion protein to degradation via the proteasome. On the other hand, the kinase specific activity of wild-type Cot is 3.8-fold lower than that of truncated Cot, and it appears that the last 43 amino acids of the wild-type Cot COOH-terminal domain are those responsible for this inhibition of kinase activity. In conclusion, these data demonstrate that the oncogenic activity of truncated Cot is the result of its prolonged half-life and its higher kinase specific activity with respect to wild-type Cot.