scholarly journals New Ca2+ pump isoforms generated by alternative splicing of rPMCA2 mRNA

1992 ◽  
Vol 283 (2) ◽  
pp. 355-359 ◽  
Author(s):  
H P Adamo ◽  
J T Penniston
Keyword(s):  
Amino Acids ◽  
Variable Region ◽  
Rat Plasma ◽  
Lipid Binding ◽  
Binding Domain ◽  
Kinase Substrate ◽  
Calmodulin Binding ◽  
Hypervariable Regions ◽  
C Terminus ◽  
Domain Insertion

Alternative splices capable of generating proteins with altered functions were found (by PCR) in isoform 2 of the rat plasma membrane Ca2+ pump. These splices were concentrated in two hypervariable regions. One of these regions, near the N-terminus and the lipid-binding region, could be altered by the insertion of either or both of inserts x and y. Insertion of both x and y would add 45 amino acids to the molecule. The y insert causes the appearance of a rather hydrophobic stretch of amino acids in the middle of a highly polar region. The second variable region begins in the middle of the calmodulin-binding domain. Insertion of 229 nucleotides at this point of the message converts the b form to the a form, which has an altered (and shorter) C-terminus. The calmodulin-binding domain of this shortened form has a less basic character, which would decrease the affinity for calmodulin. The b form of isoenzyme 2 contains relatively weak protein kinase A substrate sequences, such as KQNSS and KNNS. These sequences are eliminated in form a, and a strongly activated kinase substrate sequence, RRQSS, appears in a different place. Different tissues use different combinations of alternative splices, with heart and brain showing the greatest diversity.

The C-Terminus of Alpha Spectrin Binds Protein 4.2 and Is Necessary for Optimal Spectrin-Actin Binding.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 810-810 ◽  
Author(s):  
Catherine Korsgren ◽  
Connie S. Birkenmeier ◽  
Jane E. Barker ◽  
Luanne L. Peters ◽  
Samuel E. Lux
Keyword(s):  
Amino Acids ◽  
Membrane Skeleton ◽  
Binding Domain ◽  
Actin Binding ◽  
C Terminus ◽  
Actin Binding Domain ◽  
Ef Hand ◽  
Protein 4.2 ◽  
Protein 4.1R ◽  
Β Chain

Abstract The red blood cell (RBC) membrane skeleton is composed principally of short F-actin filaments crosslinked by α2β2-spectrin tetramers with the assistance of protein 4.1R. Actin and 4.1R bind to the actin-binding domain (βABD) at the N-terminus of the spectrin β-chain. The adjacent, C-terminal end of α-spectrin, contains a calmodulin-like domain (αCML, aa 2262–2418) that is also called the EF hand domain and is thought to be inert or vestigial. However, the sph1J/sph1J mouse, which has severe hereditary spherocytosis and unstable RBC membranes, makes a mutant α-spectrin that lacks the last 13 amino acids (αCMLΔC13), showing that the domain has some important function. To investigate this function we “fished” for interacting proteins using glutathione-S-transferase (GST)-fused to the CML domain—either the wildtype (αGST-CML) or sph1J (αGST-CMLΔC13). αGST-CML retrieved protein 4.2 from a 2M Tris HCl extract of spectrin-actin depleted human RBC membranes. Protein 4.2 bound αGST-CML with high affinity (Kd = 2.7 x 10−7M) but did not bind αGST-CMLΔC13. Binding was abolished by 1 mM Ca2+, which converts the CML domain to the liganded conformation. The binding site on protein 4.2 localized, at least partly, to amino acids 411–492. Because red cells lacking protein 4.2 are not as severely affected as sph1J/sph1J RBCs, we also tested the effect of the αCMLΔC13 mutation on spectrin-actin binding. A minispectrin was prepared containing the actin-binding domain plus the first four spectrin repeats of the β-chain, combined with the CML domain (±ΔC13) and the last four repeats of the α-chain. The normal and mutant minispectrins were incubated with protein 4.1R, F-actin, or both proteins. The results were striking. The minispectrin containing the normal CML domain bound actin in the presence of protein 4.1R, but the minispectrin containing the mutant CML domain did not. Similarly, the mutant minispectrin was defective in its ability to bind 125I-4.1R in the presence of a constant amount of F-actin. However, the mutation did not affect binding of the minispectrin to protein 4.1R in the absence of actin. We have not yet tested whether protein 4.2 or Ca2+ modulate the effects of the CML domain on spectrin-actin binding. In summary, these experiments clearly show that the calmodulin-like (EF hand) domain of α-spectrin, which was previously considered inert, binds protein 4.2 and also contributes to spectrin-actin binding in the presence of protein 4.1R. Further experiments will be needed to determine whether the CML domain binds actin directly or strengthens the binding of the adjacent actin-binding domain.

scholarly journals Monoclonal Antibody 667 Recognizes the Variable Region A Motif of the Ecotropic Retrovirus CasBrE Envelope Glycoprotein and Inhibits Env Binding to the Viral Receptor

2003 ◽  
Vol 77 (20) ◽  
pp. 10984-10993 ◽  
Author(s):  
Hanna Dreja ◽  
Laurent Gros ◽  
Sylvie Villard ◽  
Estanislao Bachrach ◽  
Anna Oates ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Receptor Binding ◽  
Envelope Glycoprotein ◽  
Critical Role ◽  
Variable Region ◽  
Binding Domain ◽  
Target Cells ◽  
Kinetic Properties ◽  
Receptor Binding Domain

ABSTRACT Monoclonal antibody (MAb) 667 is a neutralizing mouse monoclonal antibody recognizing the envelope glycoprotein (Env) of the ecotropic neurotropic murine retrovirus CasBrE but not that of other murine retroviruses. Since 667 can be used for preclinical studies of antiviral gene therapy as well as for studying the early events of retroviral infection, we have cloned its cDNAs and molecularly characterized it in detail. Spot technique-based experiments showed that 667 recognizes a linear epitope of 12 amino acids located in the variable region A of the receptor binding domain. Alanine scanning experiments showed that six amino acids within the epitope are critical for MAb binding. One of them, D57, is not present in any other murine retroviral Env, which suggests a critical role for this residue in the selectivity of 667. MAb 667 heavy- and light-chain cDNAs were functionally characterized by transient transfection into Cos-7 cells. Enzyme-linked immunosorbent assays and Biacore studies showed that the specificities as well as the antigen-binding thermodynamic and kinetic properties of the recombinant 667 MAb (r667) produced by Cos-7 cells and those of the parental hybridoma-produced MAb (h667) were similar. However, h667 was shown to contain contaminating retroviral and/or retrovirus-like particles which interfere with both viral binding and neutralization experiments. These contaminants could successfully be removed by a stringent purification protocol. Importantly, this purified 667 could completely prevent retrovirus binding to target cells and was as efficient as the r667 MAb produced by transfected Cos-7 cells in neutralization assays. In conclusion, this study shows that the primary mechanism of virus neutralization by MAb 667 is the blocking of the retroviral receptor binding domain of CasBrE Env. In addition, the findings of this study constitute a warning against the direct use of hybridoma cell culture supernatants for studying the initial events of retroviral cell infection as well as for carrying out in vivo neutralization experiments and suggest that either recombinant antibodies or highly purified antibodies are preferable for these purposes.

scholarly journals The tau 4 activation domain of the thyroid hormone receptor is required for release of a putative corepressor(s) necessary for transcriptional silencing.

1995 ◽  
Vol 15 (1) ◽  
pp. 76-86 ◽  
Author(s):  
A Baniahmad ◽  
X Leng ◽  
T P Burris ◽  
S Y Tsai ◽  
M J Tsai ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thyroid Hormone ◽  
Ligand Binding ◽  
Hormone Receptor ◽  
Thyroid Hormone Receptor ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Activation Domain ◽  
C Terminus

The C terminus of nuclear hormone receptors is a complex structure that contains multiple functions. We are interested in the mechanism by which thyroid hormone converts its receptor from a transcriptional silencer to an activator of transcription. Both regulatory functions are localized in the ligand binding domain of this receptor superfamily member. In this study, we have identified and characterized several functional domains within the ligand binding domain of the human thyroid hormone receptor (TR beta) conferring transactivation. Interestingly, these domains are localized adjacent to hormone binding sites. One activation domain, designated tau 4, is only 17 amino acids in length and is localized at the extreme C terminus of TR. Deletion of six amino acids of tau 4 resulted in a receptor that could still bind hormone but acted as a constitutive silencer, indicating that tau 4 is required for both transactivation and relief of the silencing functions. In addition, we performed in vivo competition experiments, the results of which suggest that in the absence of tau 4 or hormone, TR is bound by a corepressor protein(s) and that one role of hormone is to release corepressor from the receptor. We propose a general model in which the role of hormone is to induce a conformational change in the receptor that subsequently affects the action of tau 4, leading to both relief of silencing and transcriptional activation.

scholarly journals Two Distinct Domains within CIITA Mediate Self-Association: Involvement of the GTP-Binding and Leucine-Rich Repeat Domains

2001 ◽  
Vol 21 (9) ◽  
pp. 3001-3011 ◽  
Author(s):  
Michael W. Linhoff ◽  
Jonathan A. Harton ◽  
Drew E. Cressman ◽  
Brian K. Martin ◽  
Jenny Pan-Yun Ting
Keyword(s):  
Amino Acids ◽  
Major Histocompatibility Complex Gene ◽  
Binding Domain ◽  
Signaling Proteins ◽  
Leucine Rich Repeat ◽  
Histocompatibility Complex ◽  
Gtp Binding ◽  
C Terminus ◽  
Self Association ◽  
First Time

ABSTRACT CIITA is the master regulator of class II major histocompatibility complex gene expression. We present evidence that CIITA can self-associate via two domains: the C terminus (amino acids 700 to 1130) and the GTP-binding domain (amino acids 336 to 702). Heterotypic and homotypic interactions are observed between these two regions. Deletions within the GTP-binding domain that reduce GTP-binding and transactivation function also reduce self-association. In addition, two leucine residues in the C-terminal leucine-rich repeat region are critical for self-association as well as function. This study reveals for the first time a complex pattern of CIITA self-association. These interactions are discussed with regard to the apoptosis signaling proteins, Apaf-1 and Nod1, which share domain arrangements similar to those of CIITA.

scholarly journals The Actin-Binding Domain of Slac2-a/Melanophilin Is Required for Melanosome Distribution in Melanocytes

2003 ◽  
Vol 23 (15) ◽  
pp. 5245-5255 ◽  
Author(s):  
Taruho S. Kuroda ◽  
Hiroyoshi Ariga ◽  
Mitsunori Fukuda
Keyword(s):  
Amino Acids ◽  
Binding Domain ◽  
Dominant Negative ◽  
Actin Binding ◽  
Dominant Negative Effect ◽  
C Terminus ◽  
Defective Mutant ◽  
Actin Binding Domain ◽  
Negative Effect ◽  
Myosin Va

ABSTRACT Melanosomes containing melanin pigments are transported from the cell body of melanocytes to the tips of their dendrites by a combination of microtubule- and actin-dependent machinery. Three proteins, Rab27A, myosin Va, and Slac2-a/melanophilin (a linker protein between Rab27A and myosin Va), are known to be essential for proper actin-based melanosome transport in melanocytes. Although Slac2-a directly interacts with Rab27A and myosin Va via its N-terminal region (amino acids 1 to 146) and the middle region (amino acids 241 to 405), respectively, the functional importance of the putative actin-binding domain of the Slac2-a C terminus (amino acids 401 to 590) in melanosome transport has never been elucidated. In this study we showed that formation of a tripartite protein complex between Rab27A, Slac2-a, and myosin Va alone is insufficient for peripheral distribution of melanosomes in melanocytes and that the C-terminal actin-binding domain of Slac2-a is also required for proper melanosome transport. When a Slac2-a deletion mutant (ΔABD) or point mutant (KA) that lacks actin-binding ability was expressed in melanocytes, the Slac2-a mutants induced melanosome accumulation in the perinuclear region, possibly by a dominant negative effect, the same as the Rab27A-binding-defective mutant of Slac2-a or the myosin Va-binding-defective mutant. Our findings indicate that Slac2-a organizes actin-based melanosome transport in cooperation with Rab27A, myosin Va, and actin.

scholarly journals Calmodulin is responsible for Ca2+-dependent regulation of TRPA1 Channels

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Raquibul Hasan ◽  
Alasdair T. S. Leeson-Payne ◽  
Jonathan H. Jaggar ◽  
Xuming Zhang
Keyword(s):  
Ion Channel ◽  
Binding Domain ◽  
Dependent Manner ◽  
Calmodulin Binding ◽  
Sensory Disorders ◽  
C Terminus

Abstract TRPA1 is a Ca2+-permeable ion channel involved in many sensory disorders such as pain, itch and neuropathy. Notably, the function of TRPA1 depends on Ca2+, with low Ca2+ potentiating and high Ca2+ inactivating TRPA1. However, it remains unknown how Ca2+ exerts such contrasting effects. Here, we show that Ca2+ regulates TRPA1 through calmodulin, which binds to TRPA1 in a Ca2+-dependent manner. Calmodulin binding enhanced TRPA1 sensitivity and Ca2+-evoked potentiation of TRPA1 at low Ca2+, but inhibited TRPA1 sensitivity and promoted TRPA1 desensitization at high Ca2+. Ca2+-dependent potentiation and inactivation of TRPA1 were selectively prevented by disrupting the interaction of the carboxy-lobe of calmodulin with a calmodulin-binding domain in the C-terminus of TRPA1. Calmodulin is thus a critical Ca2+ sensor enabling TRPA1 to respond to diverse Ca2+ signals distinctly.

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