28 research outputs found
Insights into the Function of the Unstructured N-Terminal Domain of Proteins 4.1R and 4.1G in Erythropoiesis
Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N, and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. One goal in the field is to better understand the complex regulation of cell type and isoform-specific expression of 4.1 proteins. To start answering these questions, we are studying in depth the important functions of 4.1 proteins in the organization and function of the membrane skeleton in erythrocytes. We have previously reported that the binding profiles of 4.1R80 and 4.1R135 to membrane proteins and calmodulin are very different despite the similar structure of the membrane-binding domain of 4.1G and 4.1R135. We have accumulated evidence for those differences being caused by the N-terminal 209 amino acids headpiece region (HP). Interestingly, the HP region is an unstructured domain. Here we present an overview of the differences and similarities between 4.1 isoforms and paralogs. We also discuss the biological significance of unstructured domains
Genomic Structure and Production of Variant mRNAs by Alternative Splicing in Human Lymphocyte Function-Associated Antigen-3 (LFA-3, CD58)
ヒトLFA-3(CD58)におけるゲノム構造と選択的スプライシングによるバリアントmRNAの産生
細胞表面glycoproteinであるLFA-3には,glycosyl phosphatidyl inosito1(GPI)結合型および膜貫通型など,いくつかの固有のmRNA分子種の存在が証明されている.このLFA-3の多様な転写特性とmRNA産生のメカニズムを明らかにするために,我々はゲノムクローニングに引き続き構造および機能の解析を行なった.LFA-3geneは少なくとも41.1kbの大きさを持ち,6つのエキソンにより構成されていた.2つの主なmRNA種は第5エキソンにおける選択的スプライシングacceptor selectionにより産生されており,2つのマイナーなものは,第3エキソンの選択的スプライシングdonor selectionおよびエキソンスキップによる部分的および完全な欠失により産生されていた.プライマー伸長法による解析では,一連の転写開始部位が観察され,それはTATAおよびCAT boxの欠如によるものであると考えられた.1.3kbの5\u27上流の隣接配列は配向依存性にCAT reporter遺伝子の発現を促進できたが,その転写活性は低かった.LFA-3と他の2つの関連分子CD2およびCD48の遺伝子進化がそれらのゲノム構造から推測された.Several distinct mRNA species have been demonstrated for lymphocyte function-associated antigen-3 (LFA-3, CD58), a cell-surface glycoprotein anchored either through glycosyl phosphatidyl inositol (GPI)-linkage or by a transmembrane domain. We obtained a genomic clone for the LFA-3 gene and performed structural and functional analyses. The LFA-3 gene is at least 41.1 kb in length and consists of 6 exons. Two major mRNA species are generated by alternative splicing acceptor selection in exon 5, and two other minor ones by partial or entire elimination of exon 3 as a result of alternative splice donor selection or exon-skip. Primer extension analysis revealed that transcription may be initiated at a series of sites, probably because of the absence of TATA and CAAT box sequences. A 1.3-kb 5\u27-upstream flanking region was able to drive the expression of a reporter gene in an orientation-dependent manner, but only at a low level. The evolutionary relation-ship of LFA-3 with other two related molecules, CD2 and CD48, was also discussed on the basis of genomic structure
Intracellular interactions between protein 4.1 and glycophorin C on transport vesicles, as determined by fluorescence correlation spectroscopy
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Characterization of cytoskeletal protein 4.1R interaction with NHE1 (Na+/H+ exchanger isoform 1)
NHE1 (Na(+)/H(+) exchanger isoform 1) has been reported to be hyperactive in 4.1R-null erythrocytes [Rivera, De Franceschi, Peters, Gascard, Mohandas and Brugnara (2006) Am. J. Physiol. Cell Physiol. 291, C880-C886], supporting a functional interaction between NHE1 and 4.1R. In the present paper we demonstrate that 4.1R binds directly to the NHE1cd (cytoplasmic domain of NHE1) through the interaction of an EED motif in the 4.1R FERM (4.1/ezrin/radixin/moesin) domain with two clusters of basic amino acids in the NHE1cd, K(519)R and R(556)FNKKYVKK, previously shown to mediate PIP(2) (phosphatidylinositol 4,5-bisphosphate) binding [Aharonovitz, Zaun, Balla, York, Orlowski and Grinstein (2000) J. Cell. Biol. 150, 213-224]. The affinity of this interaction (K(d) = 100-200 nM) is reduced in hypertonic and acidic conditions, demonstrating that this interaction is of an electrostatic nature. The binding affinity is also reduced upon binding of Ca(2+)/CaM (Ca(2+)-saturated calmodulin) to the 4.1R FERM domain. We propose that 4.1R regulates NHE1 activity through a direct protein-protein interaction that can be modulated by intracellular pH and Na(+) and Ca(2+) concentrations
Membrane Peroxidation and Methemoglobin Formation Are Both Necessary for Band 3 Clustering: Mechanistic Insights into Human Erythrocyte Senescence
Oxidative damage and clustering of
band 3 in the membrane have
been implicated in the removal of senescent human erythrocytes from
the circulation at the end of their 120 day life span. However, the
biochemical and mechanistic events leading to band 3 cluster formation
have yet to be fully defined. Here we show that while neither membrane
peroxidation nor methemoglobin (MetHb) formation on their own can
induce band 3 clustering in the human erythrocytes, they can do so
when acting in combination. We further show that binding of MetHb
to the cytoplasmic domain of band 3 in peroxidized, but not in untreated,
erythrocyte membranes induces cluster formation. Age-fractionated
populations of erythrocytes from normal human blood, obtained by a
density gradient procedure, have allowed us to examine a subpopulation,
highly enriched in senescent cells. We have found that band 3 clustering
is a feature of only this small fraction, amounting to ∼0.1%
of total circulating erythrocytes. These senescent cells are characterized
by an increased proportion of MetHb as a result of reduced nicotinamide
adenine dinucleotide-dependent reductase activity and accumulated
oxidative membrane damage. These findings have allowed us to establish
that the combined effects of membrane peroxidation and MetHb formation
are necessary for band 3 clustering, and this is a very late event
in erythrocyte life. A plausible mechanism for the combined effects
of membrane peroxidation and MetHb is proposed, involving high-affinity
cooperative binding of MetHb to the cytoplasmic domain of oxidized
band 3, probably because of its carbonylation, rather than other forms
of oxidative damage. This modification leads to dissociation of ankyrin
from band 3, allowing the tetrameric MetHb to cross-link the resulting
freely diffusible band 3 dimers, with formation of clusters