scholarly journals Phosphorylation of the Angiotensin II (AT1A) Receptor Carboxyl Terminus: A Role in Receptor Endocytosis

1998 ◽  
Vol 12 (10) ◽  
pp. 1513-1524 ◽  
Author(s):  
Walter G. Thomas ◽  
Thomas J. Motel ◽  
Christopher E. Kule ◽  
Vijay Karoor ◽  
Kenneth M. Baker
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Angiotensin Ii ◽  
Chinese Hamster ◽  
Carboxyl Terminus ◽  
Amino Acid Substitutions ◽  
Ang Ii ◽  
Acidic Amino Acid ◽  
Wild Type ◽  
At1a Receptor

Abstract The molecular mechanism of angiotensin II type I receptor (AT1) endocytosis is obscure, although the identification of an important serine/threonine rich region (Thr332Lys333Met334Ser335Thr336Leu337Ser338) within the carboxyl terminus of the AT1A receptor subtype suggests that phosphorylation may be involved. In this study, we examined the phosphorylation and internalization of full-length AT1A receptors and compared this to receptors with truncations and mutations of the carboxyl terminus. Epitope-tagged full-length AT1A receptors, when transiently transfected in Chinese hamster ovary (CHO)-K1 cells, displayed a basal level of phosphorylation that was significantly enhanced by angiotensin II (Ang II) stimulation. Phosphorylation of AT1A receptors was progressively reduced by serial truncation of the carboxyl terminus, and truncation to Lys325, which removed the last 34 amino acids, almost completely inhibited Ang II-stimulated 32P incorporation into the AT1A receptor. To investigate the correlation between receptor phosphorylation and endocytosis, an epitope-tagged mutant receptor was produced, in which the carboxyl-terminal residues, Thr332, Ser335, Thr336, and Ser338, previously identified as important for receptor internalization, were substituted with alanine. Compared with the wild-type receptor, this mutant displayed a clear reduction in Ang II-stimulated phosphorylation. Such a correlation was further strengthened by the novel observation that the Ang II peptide antagonist, Sar1Ile8-Ang II, which paradoxically causes internalization of wild-type AT1A receptors, also promoted their phosphorylation. In an attempt to directly relate phosphorylation of the carboxyl terminus to endocytosis, the internalization kinetics of wild-type AT1A receptors and receptors mutated within the Thr332-Ser338 region were compared. The four putative phosphorylation sites (Thr332, Ser335, Thr336, and Ser338) were substituted with either neutral [alanine (A)] or acidic amino acids [glutamic acid (E) and aspartic acid (D)], the former to prevent phosphorylation and the latter to reproduce the acidic charge created by phosphorylation. Wild-type AT1A receptors, expressed in Chinese hamster ovary cells, rapidly internalized after Ang II stimulation [t1/2 2.3 min; maximal level of internalization (Ymax) 78.2%], as did mutant receptors carrying single acidic substitutions (T332E, t1/2 2.7 min, Ymax 76.3%; S335D, t1/2 2.4 min, Ymax 76.7%; T336E, t1/2 2.5 min, Ymax 78.2%; S338D, t1/2 2.6 min, Ymax 78.4%). While acidic amino acid substitutions may simply be not as structurally disruptive as alanine mutations, we interpret the tolerance of a negative charge in this region as suggestive that phosphorylation may permit maximal internalization. Substitution of all four residues to alanine produced a receptor with markedly reduced internalization kinetics (T332A/S335A/T336A/S338A, t1/2 10.1 min, Ymax 47.9%), while endocytosis was significantly rescued in the corresponding quadruple acidic mutant (T332E/S335D/T336E/S338D, t1/2 6.4 min, Ymax 53.4%). Double mutation of S335 and T336 to alanine also diminished the rate and extent of endocytosis (S335A/T336A, 3.9 min, Ymax 69.3%), while the analogous double acidic mutant displayed wild type-like endocytotic parameters (S335D/T336E, t1/2 2.6 min, Ymax 77.5%). Based on the apparent rescue of internalization by acidic amino acid substitutions in a region that we have identified as a site of Ang II-induced phosphorylation, we conclude that maximal endocytosis of the AT1A receptor requires phosphorylation within this serine/threonine-rich segment of the carboxyl terminus.

scholarly journals Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo.

1997 ◽  
Vol 17 (1) ◽  
pp. 115-122 ◽  
Author(s):  
M B Sainz ◽  
S A Goff ◽  
V L Chandler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transcriptional Activation ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Activation Domain ◽  
Hydrophobic Residues ◽  
Transcriptional Activation Domain ◽  
Acidic Amino Acids

C1 is a transcriptional activator of genes encoding biosynthetic enzymes of the maize anthocyanin pigment pathway. C1 has an amino terminus homologous to Myb DNA-binding domains and an acidic carboxyl terminus that is a transcriptional activation domain in maize and yeast cells. To identify amino acids critical for transcriptional activation, an extensive random mutagenesis of the C1 carboxyl terminus was done. The C1 activation domain is remarkably tolerant of amino acid substitutions, as changes at 34 residues had little or no effect on transcriptional activity. These changes include introduction of helix-incompatible amino acids throughout the C1 activation domain and alteration of most single acidic amino acids, suggesting that a previously postulated amphipathic alpha-helix is not required for activation. Substitutions at two positions revealed amino acids important for transcriptional activation. Replacement of leucine 253 with a proline or glutamine resulted in approximately 10% of wild-type transcriptional activation. Leucine 253 is in a region of C1 in which several hydrophobic residues align with residues important for transcriptional activation by the herpes simplex virus VP16 protein. However, changes at all other hydrophobic residues in C1 indicate that none are critical for C1 transcriptional activation. The other important amino acid in C1 is aspartate 262, as a change to valine resulted in only 24% of wild-type transcriptional activation. Comparison of our C1 results with those from VP16 reveal substantial differences in which amino acids are required for transcriptional activation in vivo by these two acidic activation domains.

B-Cell Epitope Mapping of Monoclonal Anti-Factor VIII C2 Domain Inhibitors: Identification of Amino Acids That Contribute Significant Antigen-Antibody Binding Affinity.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1211-1211
Author(s):  
Jasper C. Lin ◽  
Jason T. Schuman ◽  
Shannon L. Meeks ◽  
John F. Healey ◽  
Arthur R. Thompson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Factor Viii ◽  
Dissociation Constants ◽  
Antibody Binding ◽  
Amino Acid Substitutions ◽  
C2 Domain ◽  
Wild Type ◽  
Mutant Proteins

Abstract The most troublesome clinical complication that can afflict hemophilia A patients who receive factor VIII (FVIII) infusions as replacement therapy is the development of an anti-FVIII immune response, in which antibodies bind to functionally important FVIII surfaces, thereby blocking the pro-coagulant function of this important plasma protein cofactor. These antibodies, commonly referred to as “FVIII inhibitors”, bind primarily to the FVIII A2 and C2 domains and to the C-terminal region of the C1 domain, and inhibitors mapping to other regions have also been seen. There are multiple epitopes on the FVIII C2 domain, reflecting both its immunogenicity/antigenicity and its diverse roles in mediating interactions between FVIII and other molecules. For example, the C2 domain is essential for binding of FVIII to its carrier protein von Willebrand factor (VWF). Proteolytic activation to FVIIIa causes its release from VWF and subsequent binding to negatively charged membrane surfaces, e.g. on activated platelets, whereupon a region that overlaps the VWF binding site contacts the membrane. The C2 domain also interacts with thrombin and factor Xa, which both can activate FVIII. To better understand the basis for FVIII inhibition, and to better delineate functionally important FVIII surfaces, a panel of 56 murine anti-C2 monoclonal antibodies was generated. Competition ELISAs and functional assays were used to classify the antibodies into five groups corresponding to distinct regions on the C2 surface, which comprised a larger number of distinct epitopes (Meeks et al., Blood110, 4234–42, 2007). The present study is a high-resolution mapping of the epitopes recognized by six representative antibodies (2-77, 2-117, 3D12, 3E6, I109 and I54) using surface plasmon resonance (SPR). Each antibody was immobilized covalently via amine coupling to a CM5 chip or was captured by a rat anti-mouse IgG attached covalently to a CM5 chip. Referring to the FVIII C2 domain crystal structure (Pratt et al., Nature402, 439–42, 1999), surface-exposed amino acids were selected for mutagenesis using the Stratagene Quik-Change system, and C2 constructs with single substitutions to alanine or amino acids that were structurally similar to the wild-type residues were generated. Forty-five of these proteins were expressed in E. coli and purified; their purity and structural integrity were confirmed by SDS-PAGE and Western blot analysis. The on- and off-rates for binding of these proteins to the six monoclonal antibodies were determined using a Biacore T100 instrument. Mutations that affected binding significantly were analyzed by measuring association and dissociation constants over a temperature gradient (10–40°C), yielding estimates of changes in antibody-binding energy (ΔΔGº) of these mutant proteins compared to wild-type C2. Van’t Hoff analysis was carried out to determine the relative contributions of enthalpy and entropy to the binding energies. Interestingly, C2 binding to each antibody was abrogated by 1–5 of the 45 amino acid substitutions tested. Each of these C2 mutants bound to other antibodies with affinities similar to that of wild-type C2, indicating that this was not an artifact due to protein misfolding. The following substitutions resulted in little or no binding, as evidenced by a completely abated signal (very low Rmax compared to the wild-type C2 protein): L2273A (2-77, 2-117), R2220A (3D12, I109), Q2231A (I54) and T2272A (I109). Additional mutant proteins with reduced binding to inhibitor(s) displayed markedly higher dissociation constants and sometimes less pronounced differences in association constants compared to wild-type C2. Although several FVIII residues contributed to more than one epitope, each antibody had a unique epitope map profile. Our results suggest that a limited number of amino acid substitutions could produce a modified FVIII protein capable of eluding immunodominant inhibitors. This approach could eventually find clinical application as a novel strategy to achieve hemostasis in patients with an established FVIII inhibitor.

scholarly journals Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

2012 ◽  
Vol 443 (1) ◽  
pp. 249-258 ◽  
Author(s):  
Katherine J. Massey ◽  
Quanwen Li ◽  
Noreen F. Rossi ◽  
Raymond R. Mattingly ◽  
Douglas R. Yingst
Keyword(s):  
Angiotensin Ii ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Second Phase ◽  
Ang Ii ◽  
Wild Type ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Two Phases ◽  
At1a Receptor

Kidney plasma membranes, which contain a single α-1 isoform of Na+/K+-ATPase, simultaneously contain two sub-conformations of E2P, differing in their rate of digoxin release in response to Na+ and ATP. Treating cells with Ang II (angiotensin II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. In the present study we tested whether Ang II regulates release by increasing phosphorylation at Ser11/Ser18 and Ser938. Opossum kidney cells co-expressing the AT1a receptor and either α-1.wild-type, α-1.S11A/S18A or α-1.S938A were treated with or without 10 nM Ang II for 5 min, increasing phosphorylation at the three sites. Na+/K+-ATPase was bound to digoxin-affinity columns in the presence of Na+, ATP and Mg2+. A solution containing 30 mM NaCl and 3 mM ATP eluted ~20% of bound untreated Na+/K+-ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in α-1.wild-type and α-1.S938A, but not α-1.S11A/S18A cells. Another 50% of bound Na+/K+-ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150 mM NaCl and 3 mM ATP. Ang II increased the initial rate and slowed the second phase in α-1.wild-type, but not α-1.S938A, cells. Thus Ang II changes the conformation of two forms of EP2 via differential phosphorylation.

scholarly journals Proper Processing of Avian Sarcoma/Leukosis Virus Capsid Proteins Is Required for Infectivity

2001 ◽  
Vol 75 (13) ◽  
pp. 6016-6021 ◽  
Author(s):  
Yan Xiang ◽  
Rebekah Thorick ◽  
Marcy L. Vana ◽  
Rebecca Craven ◽  
Jonathan Leis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Virus Assembly ◽  
Leukemia Virus ◽  
Carboxyl Terminus ◽  
Amino Terminus ◽  
Amino Acid Substitutions ◽  
Gag Polyprotein ◽  
Initial Cleavage ◽  
Avian Sarcoma

ABSTRACT The formation of the mature carboxyl terminus of CA in avian sarcoma/leukemia virus is the result of a sequence of cleavage events at three PR sites that lie between CA and NC in the Gag polyprotein. The initial cleavage forms the amino terminus of the NC protein and releases an immature CA, named CA1, with a spacer peptide at its carboxyl terminus. Cleavage of either 9 or 12 amino acids from the carboxyl terminus creates two mature CA species, named CA2 and CA3, that can be detected in avian sarcoma/leukemia virus (R. B. Pepinsky, I. A. Papayannopoulos, E. P. Chow, N. K. Krishna, R. C. Craven, and V. M. Vogt, J. Virol. 69:6430–6438, 1995). To study the importance of each of the three CA proteins, we introduced amino acid substitutions into each CA cleavage junction and studied their effects on CA processing as well as virus assembly and infectivity. Preventing cleavage at any of the three sites produced noninfectious virus. In contrast, a mutant in which cleavage at site 1 was enhanced so that particles contained CA2 and CA3 but little detectable CA1 was infectious. These results support the idea that infectivity of the virus is closely linked to proper processing of the carboxyl terminus to form two mature CA proteins.

Effects of amino acid substitutions at the active site in Escherichia coli beta-galactosidase.

Genetics ◽  
1988 ◽  
Vol 120 (3) ◽  
pp. 637-644
Author(s):  
C G Cupples ◽  
J H Miller
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Lacz Gene ◽  
Wild Type ◽  
Mutant Proteins ◽  
Beta Galactosidase

Abstract Forty-nine amino acid substitutions were made at four positions in the Escherichia coli enzyme beta-galactosidase; three of the four targeted amino acids are thought to be part of the active site. Many of the substitutions were made by converting the appropriate codon in lacZ to an amber codon, and using one of 12 suppressor strains to introduce the replacement amino acid. Glu-461 and Tyr-503 were replaced, independently, with 13 amino acids. All 26 of the strains containing mutant enzymes are Lac-. Enzyme activity is reduced to less than 10% of wild type by substitutions at Glu-461 and to less than 1% of wild type by substitutions at Tyr-503. Many of the mutant enzymes have less than 0.1% wild-type activity. His-464 and Met-3 were replaced with 11 and 12 amino acids, respectively. Strains containing any one of these mutant proteins are Lac+. The results support previous evidence that Glu-461 and Tyr-503 are essential for catalysis, and suggest that His-464 is not part of the active site. Site-directed mutagenesis was facilitated by construction of an f1 bacteriophage containing the complete lacZ gene on a single EcoRI fragment.

scholarly journals An immunodominant epitope present in multiple class I MHC molecules and recognized by cytotoxic T lymphocytes.

1988 ◽  
Vol 168 (1) ◽  
pp. 307-324 ◽  
Author(s):  
D W Mann ◽  
E McLaughlin-Taylor ◽  
R B Wallace ◽  
J Forman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Class I ◽  
Target Cells ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Class I Mhc ◽  
Alpha Helices ◽  
Mhc Molecules ◽  
Natural Tolerance

CTL derived from (C3H x B6.K1)F1 animals were sensitized against L cells that express the transfected gene product Q10d/Ld. These CTL were highly crossreactive against three other class I molecules, H-2Kbm1, H-2Ld, and H-2Kd. In an attempt to define this crossreactive epitope it was noted that between 25 and 39% of amino acids in the alpha helices and central beta strands of these three molecules vary from Q10d. These amino acids represent residues that have been proposed to potentially interact with a peptide antigen or TCR (21). However, all four molecules share the amino acid tyrosine at positions 155 and 156. Additionally, Q10d, H-2Kbm1, and H-2Ld share alanine at position 152, while H-2Kd has an aspartic acid. We showed that these residues were important in controlling this epitope by the finding that anti-Q10d CTL did not recognize H-2Kbm1 revertant molecules that had either the position 152 alanine changed back to the wild-type H-2Kb residue (glutamic acid) or position 155 and 156 tyrosines changed back to wild-type residues arginine and leucine. Further evidence that these molecules share a crossreactive epitope was noted by the failure of (C3H x H-2Kbm1)F1 animals to generate CTL that recognized H-2Ld or H-2Kd, and the inability of (C3H x BALB/c)F1 animals to generate CTL reactive against H-2Kbm1. CTL from these mice were still able to recognize Q10d/Ld indicating that other epitopes could be detected if natural tolerance prevented recognition of the crossreactive epitope. To further define the epitope, CTL clones were generated against Q10d/Ld and maintained on either H-2Kbm1 or BALB/c feeder cells. In addition to testing these clones on the target cells described above, mutant molecules derived from H-2Ld, which have amino acid substitutions in their alpha 1 domain, were analyzed. It was noted that some anti-Q10 clones that did not crossreact on H-2Ld did react against H-2Ld mutant antigens that had H-2Dd amino acid substitutions in the alpha 1 domain at positions 63, 65, 66, and 70. Other clones had differential reactivities on these H-2Ld mutants further substantiating that alpha 1 domain amino acids play a role in controlling the expression of the crossreactive epitope. Thus, four class I molecules with multiple amino acid differences in their alpha 1 and alpha 2 domains share a crossreactive epitope readily recognized by alloreactive CTL.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

1992 ◽  
Vol 68 (06) ◽  
pp. 672-677 ◽  
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Half Life ◽  
Tissue Type ◽  
Clot Lysis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

Lipid signal transduction pathways in angiotensin II type 1 receptor-transfected fibroblasts

1995 ◽  
Vol 269 (2) ◽  
pp. C435-C442 ◽  
Author(s):  
Y. Wen ◽  
M. C. Cabot ◽  
E. Clauser ◽  
S. L. Bursten ◽  
J. L. Nadler
Keyword(s):  
Signal Transduction ◽  
Angiotensin Ii ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Signal Transduction Pathways ◽  
Ang Ii ◽  
Vascular Type ◽  
Lipid Signal ◽  
Fibroblast Line

A stable Chinese hamster ovary fibroblast line expressing the rat vascular type 1a angiotensin II (ANG II) receptor was used to study the lipid-derived signal transduction pathways elicited by type 1a ANG II receptor activation. ANG II caused a biphasic and dose-dependent increase in diacylglycerol (DAG) accumulation with an initial peak at 15 s (181 +/- 11% of control, P < 0.02) and a second sustained peak at 5-10 min (214 +/- 10% of control, P < 0.02). The late DAG peak was derived from phosphatidylcholine (PC), and the formation was blocked by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ANG II also increased phosphatidic acid (PA) production nearly fourfold by 7.5 min. In the presence of ethanol, ANG II markedly increased phosphatidylethanol (PEt) formation, indicating activation of phospholipase D (PLD). ANG II was shown to increase the mass of three separate PA species, one of which apparently originated from DAG kinase action on PC-phospholipase C (PLC)-produced DAG, providing evidence for PC-PLC activity. ANG II also formed a third PA species, which originated neither from PLD nor from DAG kinase. These results demonstrate that multiple lipid signals propagated via collateral stimulation of PLC and PLD are generated by specific activation of the vascular type 1a ANG II receptor.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

Sign in / Sign up

Export Citation Format

Share Document