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PubMedDate not supplied

Human islets of Langerhans express the delta(C) isoform of calcium/calmodulin-dependent protein kinase II.

BACKGROUND: There is considerable evidence that calcium/calmodulin-dependent protein kinase II (CaM kinase II) plays a key role in insulin secretion and the enzyme provides a candidate gene for Type 2 diabetes. Since several isoforms of the enzyme exist, it is essential to define which are expressed by the beta-cell. METHODS: A human islet cDNA library in lambdaZAPII was screened with a probe for the 5'-end of human gamma CaM kinase II. Since this region is very homologous between the different isoforms, it is expected that isoforms other than gamma would be detected. From each of the six positive clones obtained, DNA was prepared and subjected to PCR using primers spanning the variable region in which the main variability of CaM kinase II isoforms resides. PCR products were purified and sequenced in both directions. The beta-cell line MIN6 was screened for CaM kinase II delta by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Western blotting. RESULTS: The sequences of five of the human islet PCR products indicated that the clones corresponded to the gamma(B) isoform whose expression in human islets we have previously documented. The other PCR product, however, gave a sequence containing the variable domains II and VII characteristic of CaM kinase II delta. This sequence and the absence of other domains in this region identified the clone as CaM kinase II delta(C). The expression of CaM kinase II delta in MIN6 beta-cells was confirmed by RT-PCR and by Western blotting. CONCLUSIONS: Human islets of Langerhans express the delta(C) isoform of CaM kinase II.

Amino Acid Sequence↗
PubMed2000

Expression of vascular endothelial growth factor isoforms and their receptors Flt-1, KDR, and neuropilin-1 in synovial tissues of rheumatoid arthritis.

Angiogenesis is an indispensable process in the chronic proliferative synovitis and pannus formation of rheumatoid arthritis (RA). This study examined the expression of vascular endothelial growth factor (VEGF) isoforms and VEGF receptors, Flt-1, KDR and neuropilin-1, in RA and osteoarthritis (OA) synovia, and studied the relationship between their expression and the synovial angiogenesis. By RT-PCR analysis, the isoform VEGF(121) was constitutively expressed in all the RA (17/17 patients) and OA (8/8 patients) synovia. In contrast, the expression of the isoform VEGF(165) was observed in 41% of the RA synovia (7/17 patients), but was undetectable in the OA samples (0/8 patients). The receptor Flt-1 was almost constitutively expressed in RA (15/17 patients) and OA (8/8 patients) synovia, while the expression of KDR was detected in the synovia of six RA patients (6/17 patients; 35%) but none of the OA patients (0/8 patients). The expression of neuropilin-1, an isoform-specific receptor for VEGF(165) which enhances the binding of VEGF(165) to KDR, was also up-regulated in the same RA synovia that expressed KDR. Furthermore, there was a close correlation between the expression of isoform VEGF(165) and that of its receptors KDR and neuropilin-1. Morphometric analysis demonstrated that the vascular density is significantly higher in the RA synovial tissues with expression of VEGF(165), KDR, and neuropilin-1 than in those without their expression (p<0.01). In situ hybridization and immunohistochemical studies indicated that the cells expressing VEGF are macrophage-like synovial lining cells and spindle-shaped cells in the sublining cell layer. These results suggest that the selective up-regulation of the isoform VEGF(165) and its signalling via KDR and neuropilin-1 play an important role in the synovial angiogenesis which occurs in RA.

Adult↗
PubMed2000

Protein kinase C isoforms involved in regulation of cell shape and locomotion of human fibrosarcoma HT1080 cells.

The role of protein kinase C (PKC) isoforms in the regulation of cell shape [switch between fibroblast-like and crescent shape (CS)] and of locomotion of human fibrosarcoma HT1080 cells has been investigated. The PKC activator phorbol myristate acetate (PMA) induced the transition of elongated fibroblast-like cells into CS cells and stimulated locomotion. Both responses to PMA were inhibited by the PKC inhibitor Ro 31-8220. Analysis of the time course showed that stimulation of shape changes (formation of CS cells) and locomotor activity (increase in the proportion and speed of locomoting cells) was maximal in the early phase of the response (up to 2.5 hr) and significantly decreased later (15 to 21 hr). CS formation and stimulated locomotion correlated closely with a marked redistribution from the cytosol to the membrane of PKC isoforms alpha, beta1 and gamma in the early phase (0.5 to 2 hr) following activation with PMA. The subsequent reduction of the proportion of CS cells and of cell locomotion correlated with down-regulation of these isoforms in the second phase (16 to 21 hr). In contrast, the total amount and distribution of PKC beta2 remained almost unchanged with 10(-8) M PMA up to 21 hr. Furthermore, changes in shape and locomotion did not correlate with the responses of PKC delta to PMA. Inhibition of PMA-stimulated locomotion by the more specific inhibitor Gö 6976 is consistent with a role of PKC alpha and beta1 in this response. Ro 31-8220 alone induced a moderate down-regulation of PKC isoforms alpha and delta, but it also inhibited the more pronounced down-regulation of these isoforms by PMA. Our results indicate that activation of PKC isoforms alpha, gamma and beta1, but not beta2 or delta, stimulates locomotion and formation of CS cells in human fibrosarcoma HT1080 cells.

Carbazoles↗
PubMed1995

Analysis of transcriptional isoforms of collagen types IX, II, and I in the developing avian cornea by competitive polymerase chain reaction.

The genes for the alpha 1(IX), alpha 1(II), and alpha 2(I) collagen chains can give rise to different isoforms of mRNA, generated by alternative promotor usage [for alpha 1(IX) and alpha 2(I)] or alternative splicing [for alpha 1(II)]. In this study, we employed competitive reverse transcriptase PCR to quantitate the amounts of transcriptional isoforms for these genes in the embryonic avian cornea from its inception (about 3 1/2 days of development) to 11 days. In order to compare values at different time points, the results were normalized to those obtained for the "housekeeping" enzyme, glycerol-3-phosphate dehydrogenase (G3PDH). These values were compared to those obtained from other tissues (anterior optic cup and cartilage) that synthesize different combinations of the collagen isoforms. We found that, in the cornea, transcripts from the upstream promotor of alpha 1(IX) collagen (termed "long IX") were predominant at stage 18-20 (about 3 1/2 days), but then fell rapidly, and remained at a low level. By 5 days (just before stromal swelling) the major mRNA isoform of alpha 1(IX) was from the downstream promoter (termed "short IX"). The relative amount of transcript for the short form of type IX collagen rose to a peak at about 6 days of development, and then declined. Throughout this period, the predominant transcriptional isoform of the collagen type II gene was IIA (i.e., containing the alternatively spliced exon 2). This indicates that the molecules of type II collagen that are assembled into heterotypic fibrils with type I collagen possess, at least transiently, an amino-terminal globular domain similar to that found in collagen types I, III, and V. For type I, the "bone/tendon" mRNA isoform of the alpha 2(I) collagen gene was predominant; transcripts from the downstream promotor were at basal levels. In other tissues expressing collagen types IX and II, long IX was expressed predominantly with the IIA form in the anterior optic cup at stage 22/23; in 14 1/2 day cartilage, long IX was expressed predominantly along with the IIB form of alpha 1(II). The downstream transcript of the alpha 2(I) gene (Icart) was found at high levels only in cartilage.

Animals↗
PubMedDate not supplied

Quantification of isoforms of plasma MM creatine kinase (CK) with an immunoblot procedure.

The MM isoenzyme of creatine kinase, a dimer composed of two M ("muscle type") subunits, is found in myocardium, where it constitutes 85% of tissue CK, and in skeletal muscle, where it constitutes virtually 100%, as well as in other tissues. The tissue form is designated MMA. When MMA circulates in plasma, it undergoes stepwise, post-translational modification, mediated by proteolytic enzymes in plasma and giving rise to isoforms called MMB and MMC, which lack carboxy terminal lysine on one or two subunits, respectively. We have shown previously that changes with time in plasma profiles of MM creatine kinase (CK) isoforms in dogs reflect myocardial infarction within 1 hour after the onset of coronary occlusion and permit noninvasive detection of reperfusion within 30 minutes after release of an occlusive coronary arterial ligature. However, analysis of MM CK isoforms in plasma from patients has been hampered by the lack of availability of quantitative as opposed to qualitative methods. This study was performed to develop and validate an assay with the sensitivity and specificity needed for accurate quantification of MM CK isoforms in samples of plasma from patients. A rapid assay procedure will be required ultimately for prospective, clinical use. However, as a first step and for use in development and standardization of rapid assays, a procedure is needed for accurate qualification of isoforms even if its implementation is laborious and slow. The isoform composition of normal plasma was found to comprise 32.0% MMA, 34.9% MMB, and 32.7% MMC.(ABSTRACT TRUNCATED AT 250 WORDS)

Creatine Kinase↗
PubMed1994

Differential regulation of skeletal muscle myosin-II and brush border myosin-I enzymology and mechanochemistry by bacterially produced tropomyosin isoforms.

In this report, we have compared the physical properties and actin-binding characteristics of several bacterially produced nonmuscle and striated muscle tropomyosins, and we have examined the effects of these isoforms on the interactions of actin with two structurally distinct classes of myosin: striated muscle myosin-II and brush border (BB) myosin-I. All of the bacterially produced nonmuscle tropomyosins bind to F-actin with the expected stoichiometry and with affinities comparable to that of a tissue produced alpha-tropomyosin, although the striated muscle tropomyosin CTm7 has a lower affinity for F-actin than a tissue-purified striated muscle alpha tropomyosin. The bacterially produced isoforms also protect F-actin from severing by villin as effectively as tissue-purified striated muscle alpha-tropomyosin. The bacterially produced 284 amino acid striated muscle tropomyosin isoform CTm7, the 284 amino acid nonmuscle tropomyosin isoform CTm4, and two chimeric tropomyosins (CTm47 and CTm74) all inhibit the actin-activated MgATPase activity of muscle myosin S1 by approximately 70-85%, comparable to the inhibition seen with tissue-purified striated muscle alpha tropomyosin. The 248 amino acid tropomyosin XTm4 stimulated the actin-activated MgATPase activity of muscle myosin S1 approximately two- to threefold. The in vitro sliding of actin filaments translocated by muscle myosin-II (2.4 microns/sec at 19 degrees C, 5.0 microns/s at 24 degrees C) increased 25-65% in the presence of XTm4. Tropomyosins CTm4, CTm7, CTm47, and CTm74 had no detectable effect on myosin-II motility. The actin-activated MgATPase activity of BB myosin-I was inhibited 75-90% by all of the tropomyosin isoforms tested, including the 248 amino acid tropomyosin XTm4. BB myosin-I motility (50 nm/s) was completely inhibited by both the 248 and 284 amino acid tropomyosins. These results demonstrate that bacterially produced tropomyosins can differentially regulate myosin enzymology and mechanochemistry, and suggest a role for tropomyosin in the coordinated regulation of myosin isoforms in vivo.

Actins↗
PubMed2002

Cell-specific expression of plasma membrane calcium ATPase isoforms in retinal neurons.

Ca(2+) extrusion by high-affinity plasma membrane calcium ATPases (PMCAs) is a principal mechanism for the clearance of Ca(2+) from the cytosol. The PMCA family consists of four isoforms (PMCA1-4). Little is known about the selective expression of these isoforms in brain tissues or about the physiological function conferred upon neurons by any given isoform. We investigated the cellular and subcellular distribution of PMCA isoforms in a mammalian retina. Mouse photoreceptors, cone bipolar cells and horizontal cells, which respond to light with a graded polarization, express isoform 1 (PMCA1) of the PMCA family. PMCA2 is localized to rod bipolar cells, horizontal cells, amacrine cells, and ganglion cells, and PMCA3 is predominantly expressed in spiking neurons, including both amacrine and ganglion cells but is also found in horizontal cells. PMCA4 was found to be selectively expressed in both synaptic layers. Optical measurements of Ca(2+) clearance showed that PMCAs mediate Ca(2+) extrusion in both rod and cone bipolar cells. In addition, we found that rod bipolar cells, but not cone bipolar cells possess a prominent Na(+)/Ca(2+) exchange mechanism. We conclude that PMCA isoforms are selectively expressed in retinal neurons and that processes of Ca(2+) clearance are different in rod and cone bipolar cells.

Amacrine Cells↗
PubMed1992

Isoform-specific associations of CD45 with accessory molecules in human T lymphocytes.

Association of CD45 with surface molecules was investigated in human T lymphocytes by co-capping. CD45 appeared to be associated with the CD3/T cell receptor complex and with CD4 or CD8 molecules in memory, but not in naive T cells, as previously reported in the mouse. Associations of CD45 isoforms with accessory molecules were then identified with seven anti-CD45R monoclonal antibodies (mAb). An isoform-specific association pattern was observed: CD2 co-capped with CD45 molecules recognized by UCHL1 mAb (CD45R0). LFA-1 with molecules bound by 2H4 mAb (CD45RA), and both CD4 and CD8 with molecules reacting with MCA.347 mAb (whose isoform specificity was not known). Further information on the CD45 isoform(s) associated to CD4 and CD8 was sought by assessing the isoform specificity of MCA.347. Cross-competition experiments showed that it reacts with an epitope clearly different from those recognized by 2H4 and UCHL1, and only partially overlapping the PD7/26 epitope (CD45RB). Moreover, the competition between MCA.347 and PD7/26 was maximal in naive T cells and minimal both in memory T cells and in a subset expressing CD11b, a marker of granular lymphocytes. Immunoprecipitation experiments showed that MCA.347 binds to CD45 molecules with a molecular mass of 220, 205 and 190 kDa, the 190-kDa molecules not being recognized by 2H4, PD7/26 or UCHL1. These data indicate that MCA.347 recognizes amino acid sequences different from those coded by the exon A or B of the gene, and not expressed by CD45R0, suggesting that it binds to sequences coded by the exon C. In conclusion, this work shows that in human T cells different CD45 isoforms are associated to different surface molecules: LFA-1 is associated to CD45RA, CD2 to CD45R0 and CD4 and CD8 presumably to CD45RC. This peculiar behavior of CD45 suggests that it may play a crucial role in lymphocyte activation, probably by modulating the signals delivered to the cell by different receptor systems.

Antibodies, Monoclonal↗
PubMed1997

Analysis of the tyrosine phosphorylation and calcium fluxing of human CD6 isoforms with different cytoplasmatic domains.

CD6 is a cell surface glycoprotein that functions both as a co-stimulatory and adhesion receptor on T cells. Recently we have described CD6 isoforms (CD6a, b, c, d, e) that arise via alternative splicing of exons encoding the cytoplasmic region of the molecule. CD6 becomes phosphorylated on tyrosine (Tyr) residues following stimulation through the T cell receptor (TCR) complex. Since the phosphorylation of Tyr residues renders some cell surface receptors competent for interactions with proteins of intracellular signaling pathways, we wanted to determine which region(s) and residues in the cytoplasmic domain of CD6 were important for phosphorylation on Tyr residues. We engineered and stably expressed chimeric receptors that consisted of the extracellular region of mouse CD6 and the cytoplasmic regions of either naturally occurring isoforms of human CD6, truncated proteins, or point mutants. We were able to demonstrate that of the nine Tyr residues in the cytoplasmic domain of the largest isoform CD6a, the two C-terminal Tyr residues (Tyr 629/662) are critical for the phosphorylation of CD6 following TCR cross-linking. Isoform CD6e, which is missing a region that contains two proline-rich motifs, is not phosphorylated. We further analyzed the ability of the different CD6 isoforms and truncated receptors to mobilize intracellular calcium after CD6/TCR co-ligation. All CD6 isoforms, including CD6e, as well as the truncation mutant delta 555, which is missing approximately the C-terminal half of the cytoplasmic domain, are able to increase Ca2+ influx. Taken together, these results suggest that the region of CD6 which is critical for Ca2+ mobilization is located N-terminal from amino acid 555 and is therefore different from the region located at the C terminus of CD6, which is necessary for tyrosine phosphorylation.

Amino Acid Sequence↗
PubMed1995

Separation of arginase isoforms by capillary zone electrophoresis and isoelectric focusing in density gradient column.

Four major arginase isoforms, I, II, III and IV, have been detected in Evernia prunastri thallus. They differ in terms of both physical and biochemical properties. The isoelectric point (pI) of these proteins has been determined by both isoelectric focusing in density gradient column and high-performance capillary electrophoresis (HPCE). Isoelectric focusing revealed charge microheterogeneity for isoforms II and IV whereas arginases I and II had the same pI value of 5.8. HPCE separation confirmed this charge microheterogeneity for isoform IV but not for isoform III, and provided evidence of microheterogeneity for isoforms I and II. The effect of various electrolyte buffers and running conditions on the HPCE separation of arginase isoform were investigated. Addition of 0.5 mM spermidine (SPD) to the running buffer reduced the electroosmotic flow (EOF) and permitted discriminating between the native proteins and protein fragments.

Arginase↗
PubMed1998

Characterization of rat brain stathmin isoforms by two-dimensional gel electrophoresis-matrix assisted laser desorption/ionization and electrospray ionization-ion trap mass spectrometry.

Stathmin is a regulatory phosphoprotein that is a target for both cell cycle and cell surface receptor-regulated phosphorylation events. There are at least 14 isoforms of stathmin that migrate on two-dimensional electrophoresis (2-DE): two unphosphorylated, and 12 increasingly phosphorylated proteins. Following extracellular stimuli, stathmin is phosphorylated on four serines (Ser16, Ser25, Ser38, and Ser63) by several kinases, such as mitogen-activated protein (MAP), cdc2 kinase, protein kinase A, and Ca2+/calmodulin-dependent kinase-Gr. While all forms of stathmin are derived from the same protein encoded by a single mRNA, the precise nature of the post-translational modifications has not been clear. In this study we have characterized three rat brain stathmin isoforms, #1, #3 and #4, which electrophorese on 2-DE with apparent molecular weight (Mr)/isoelectric point (pI) values of 15,500/6.2, 15,000/6.1, and 15,000/6.0, respectively. The phosphorylation status of these isoforms was determined using a combination of peptide mapping, matrix-assisted laser desorption/ionization mass spectrometry and electrospray-ionization ion trap mass spectrometry. Stathmin isoform #1 was not phosphorylated, stathmin isoform #3 was phosphorylated on Ser38 only, and stathmin isoform #4 was phosphorylated on Ser38; however, the phosphorylation status of Ser63 could not be determined. In addition, three proteins which electrophorese near stathmin were identified in order to more accurately define the Mr/pI locus of this region of the 2-DE gel map. These include: phosphatidyl ethanolamine binding protein (Mr approximately 18,000/pI 6.0), synuclein forms 2 and 3 (Mr approximately 14,000/pI 5.4), and synuclein form 2 (Mr approximately 15,000/pI 5.0).

Amino Acid Sequence↗
PubMed1989

Expression of Na,K-ATPase alpha subunit isoforms in the human ciliary body and cultured ciliary epithelial cells.

We have analyzed the expression of Na,K-ATPase alpha subunit isoforms in the transporting ciliary processes of the human eye and in cultured cells derived from non-pigmented (NPE) and pigmented (PE) ciliary epithelium. Northern hybridization analysis shows that the mRNAs encoding all the three distinct forms of Na,K-ATPase alpha subunit [alpha 1, alpha 2, and alpha 3] are expressed in the human ciliary processes in vivo. Immunohistochemical analysis using antibodies specific for each of the three alpha subunit isoforms confirms that these polypeptides are present in the microsomal fraction from the human ciliary processes. The monoclonal antibody McB2, which is specific to the Na,K-ATPase alpha 2 subunit isoform, has been found to decorate specifically the basolateral membrane domains of NPE cells but not of the PE cells, suggesting its expression in vivo only in the ocular NPE ciliary epithelium. However, cultured cells derived from the NPE and PE layers exhibit a different pattern of expression of mRNA and protein for the Na,K-ATPase alpha subunit isoforms when compared to the tissue. Both the NPE and PE cells express alpha 1 and alpha 3 mRNA and polypeptide, whereas alpha 2 mRNA and polypeptide are undetectable in these cells. The established cell lines derived from the NPE layer express comparable levels of the alpha 1 and alpha 3 isoforms of Na,K-ATPase as detected in the primary culture. However, the established NPE cell lines are also distinguishable from the normal PE cells when analyzed by Western blot analysis with A x 2 antibodies. The results presented here clearly show that the NPE and PE cells in the ciliary body have a distinct expression of Na,K-ATPase alpha subunit isoforms as compared to cultured cells.

Blotting, Western↗
PubMed1990

Sex-dependent and independent expression of the P-glycoprotein isoforms in Chinese hamster.

P-glycoprotein (Pgp) is a small family of membrane proteins which belongs to a superfamily of energy-dependent membrane transport proteins identified in phylogenetically distant species, from bacteria to man. Among mammalian species, some of the Pgp isoforms can mediate multidrug resistance by acting as an energy-dependent drug efflux pump. However, the physiologic functions of the Pgp isoforms have not been defined. In this study we examined the expression of the three hamster Pgp isoforms in normal hamster tissues, by using isoform-specific monoclonal antibodies in a competitive immunohistochemical assay. We showed that each Pgp isoform is predominantly expressed in a small, distinct group of differentiated cells, where it is likely to function in specific secretory pathways. The expression of the Pgp isoforms appears to be tightly regulated and, at least in some cells, under complex hormonal control. Furthermore, there is a striking sex difference in Pgp content of the adrenal cortex. These findings are important for the ultimate understanding of the normal physiologic roles of the Pgp gene family members.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Expression of multiple Na+,K(+)-ATPase genes reveals a gradient of isoforms along the nonpigmented ciliary epithelium: functional implications in aqueous humor secretion.

The Na+,K(+)-ATPase alpha 1, alpha 2, and alpha 3 subunit isoforms have been shown to be differentially expressed in the nonpigmented (NPE) and pigmented (PE) cells of the ocular ciliary epithelium (CE) (Martin-Vasallo et al., J. Cell. Physiol., 141:243-252, 1989; Ghosh et al., J. Biol. Chem., 265:2935-2940, 1990). In this study we analyzed and compared the pattern of expression of the multiple Na+,K(+)-ATPase alpha (alpha 1, alpha 2, alpha 3) subunit genes with the pattern of expression of the Na+,K(+)-ATPase beta (beta 1, beta 2) subunit genes along the bovine CE. We have selected three regions in the CE, referred to as 1) the anterior region of the pars plicata, near the iris; 2) the middle region of the pars plicata; and 3) the posterior region of the pars plana, near the ora serrata. Using isoform-specific cDNA probes and antibodies for the Na+,K(+)-ATPase alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunits on Northern and Western blot analysis, we found that mRNA and polypeptides are expressed in all three CE regions with different abundance. The pattern of expression of alpha and beta isoforms detected along the NPE cell layers suggests a gradient of alpha 1, alpha 2, alpha 3, beta 1, and beta 2 mRNAs and polypeptides that correlates with decreasing Na+,K(+)-ATPase activity from the most anterior region at the pars plicata towards the posterior region at the ora serrata. We also found marked differences in the pattern of immunolocalization of Na+,K(+)-ATPase alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunit isoforms in different regions of the CE. In the anterior region, NPE cells stained intensely at the basal lateral membrane with specific monoclonal and polyclonal antibodies for each of the alpha (alpha 1, alpha 2, alpha 3) and beta (beta 1, beta 2) Na,K-ATPase isoforms. In the middle and posterior regions of the CE, NPE cells showed lower or absent levels of staining with alpha 1, alpha 2, alpha 3, and beta 1 antibodies, although staining with beta 2 was abundant. In contrast, PE cells throughout the CE were stained at the basal lateral membrane by antibodies to alpha 1 and beta 1, while no staining signals were detected with the rest of the antibodies (i.e. alpha 2, alpha 3, and beta 2). Our results support the conclusion that the three alpha and two beta isoforms of the Na+,K(+)-ATPase are differentially expressed in the two cell layers that make up the CE.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Interaction of CD44 variant isoforms with hyaluronic acid and the cytoskeleton in human prostate cancer cells.

CD44 is a glycosylated adhesion molecule which may undergo alternative splicing of 10 possible exons to generate variant isoforms. A number of CD44 variant isoforms expressed by tumor cells have been correlated with metastatic and proliferative behavior. In this study, we have characterized CD44 isoform expression on three prostate cancer cell lines: ALVA-31, PPC-1, and LNCaP. Using reverse transcriptase-polymerase chain reaction, we have found that ALVA-31 and PPC-1 cells express multiple CD44 isoforms, including CD44s (standard form), CD44E (epithelial form), and an exon 14-containing form. In addition, two smaller forms have been detected: one using an alternative donor splice site within exon 5, and a novel form omitting exon 5 entirely. The CD44 isoforms expressed by ALVA-31 and PPC-1 cells appear to be preferentially located on the cell surface. By contrast, LNCaP cells do not express any of the CD44 forms at the RNA or protein level. Both PPC-1 and ALVA-31 cells display tumorigenesis and invasiveness in nude mice, whereas LNCap cells exhibit a less malignant phenotype, suggesting a correlation between CD44 variant (CD44v) expression and aggressive prostate tumor behavior. Functional characterization reveals that CD44 mediates prostate cell adhesion to extracellular hyaluronic acid (HA). In addition, the CD44 cytoplasmic domain binds specifically to ankyrin, a membrane cytoskeletal protein. Double immunofluorescence labeling and confocal microscopic analyses indicate that HA binding induces the HA receptor (i.e., CD44) to form capped structures. Importantly, intracellular ankyrin is preferentially accumulated underneath HA receptor-capped structures. These results suggest that cytoskeletal proteins such as ankyrin are closely associated with CD44-mediated signaling events induced by HA. Finally, HA-mediated transmembrane interactions between CD44 isoforms and cytoskeletal proteins (i.e. ankyrin) may play a pivotal role in regulating tumor cell behavior during human prostate cancer development.

Ankyrins↗
PubMed2005

Thrombospondin-1 deficient mice exhibit an altered expression pattern of alternatively spliced PECAM-1 isoforms in retinal vasculature and endothelial cells.

We have previously shown that thrombosponsin-1 (TSP1) and PECAM-1 are components of a regulatory switch whose reciprocal regulation in the endothelial cells (EC) promotes an angiogenic or a differentiated, quiescent phenotype. The physiological role TSP1 plays in modulation of PECAM-1 expression and function during vascular development and angiogenesis remains largely unknown. Here we demonstrate that PECAM-1 undergoes alternative splicing in its cytoplasmic domain generating eight isoforms in the retinal vasculature of wild type and TSP1-/- mice. All PECAM-1 isoforms examined contained exon 13. The frequency of PECAM-1 isoform(s) containing exon 14 was significantly higher during early stages of retinal vascularization, which decreased during later stages of retinal vascularization in wild type mice. In contrast, the frequency of exon 14 containing PECAM-1 isoform(s) did not significantly change during retinal vascularization in TSP1-/- mice. They consistently expressed higher number of isoforms with exon 14 during later stages of retinal vascularization. The higher level of PECAM-1 isoforms with exon 14 was also observed in cultured TSP1-/- retinal EC compared to wild type retinal EC. This was consistent with increased amounts of Src and SHP-2 associated with PECAM-1, and enhanced migration and proliferation in TSP1-/- retinal EC. These data suggest PECAM-1 signaling in the endothelium is modulated by its alternative splicing during retinal vascular development and angiogenesis, which may be impacted by TSP1 expression.

Alternative Splicing↗
PubMed1995

Caldesmon and low Mr isoform of tropomyosin are localized in neuronal growth cones.

Neuronal growth cones move actively, accompanying changes in intracellular Ca2+ concentration. The movement of growth cones may partly depend on the actomyosin system, considering the presence of actin and myosin II. Yet, Ca(2+)-sensitive regulatory proteins for the actomyosin system have not been identified in growth cones. In the present study, caldesmon, an inhibitory protein on actin-myosin interaction, was detected in the growth cone fraction isolated from embryonic rat brain, using immunoblotting with the antibody to chicken gizzard caldesmon. Morphological evidence of caldesmon in growth cones of cultured rat neurons was obtained using the indirect immunofluorescence method. Since inhibition of caldesmon on actin-myosin interaction can be overcome by calmodulin and Ca2+, caldesmon may be involved in the Ca(2+)-dependent regulation in growth cone motility. Tropomyosin is another member of the actomyosin system whose function may be regulated by caldesmon in smooth and nonmuscle cells. A low Mr isoform of tropomyosin was distributed in the growth cone fraction. Using specific antibodies against tropomyosin isoforms, we further clarified morphologically that the low Mr isoform was localized in growth cones, but not the high Mr isoform. High Mr isoforms of tropomyosin were present in nonneuronal cells. Actin filaments in growth cones may be unstable, since low Mr tropomyosin binds to actin filaments with a lower affinity than high Mr isoforms. The instability of actin filaments may be suitable for the rapid movement and shape changes of growth cones.

Actins↗
PubMed2003

Cytochrome c oxidase of mammals contains a testes-specific isoform of subunit VIb--the counterpart to testes-specific cytochrome c?

Sperm motility is highly dependent on aerobic energy metabolism, of which the apparent rate-limiting step of the mitochondrial respiratory chain is catalyzed by cytochrome c oxidase (COX). COX is the only electron transport chain complex to display isoforms, consistent with its suggested rate-limiting role. Isoforms were previously described for four of the 13 subunits. We now report the discovery that COX subunit VIb displays a testes-specific isoform in human, bull, rat, and mouse (COX VIb-2). Analysis of a variety of rat and mouse tissues, including ovaries, demonstrates exclusive expression of VIb-2 in testes, whereas VIb-1 transcripts are absent in rodent testes, even at early developmental stages. In contrast, both isoforms are transcribed in human testes. In situ hybridizations with human, rat, and mouse testes sections reveal VIb-2 transcripts in all testicular cell types. Within the seminiferous tubules, VIb-1 shows stronger signals in the periphery than in the lumen. Previously, cytochrome c was the only component of the mitochondrial respiratory chain known to express a testes-specific isoform in mammals. COX subunit VIb connects the two COX monomers into the physiological dimeric form, and is the only COX subunit that, like cytochrome c, is solely located in the inter-membrane space. Significant differences between the isoform sequences, in particular changes in charged amino acids, suggest interactions with cytochrome c and sperm-specific energy requirements.

Animals↗