Interaction between CFTR and prestin (SLC26A5).

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel that is present in a variety of epithelial cell types, and usually expressed in the luminal membrane. In contrast, prestin (SLC26A5) is a voltage-dependent motor protein, which is present in the basolateral membrane of cochlear outer hair cells (OHCs), and plays an important role in the frequency selectivity and sensitivity of mammalian hearing. By using in situ hybridization and immunofluorescence, we found that both mRNA and protein of CFTR are present in OHCs, and that CFTR localizes in both the apical and the lateral membranes. CFTR was not detected in the lateral membrane of inner hair cells (IHCs) or in that of OHCs derived from prestin-knockout mice, i.e., in instances where prestin is not expressed. These results suggest that prestin may interact physically with CFTR in the lateral membrane of OHCs. Immunoprecipitation experiments confirmed a prestin-CFTR interaction. Because chloride is important for prestin function and for the efferent-mediated inhibition of cochlear output, the prestin-directed localization of CFTR to the lateral membrane of OHCs has a potential physiological significance. Aside from its role as a chloride channel, CFTR is known as a regulator of multiple protein functions, including those of the solute carrier family 26 (SLC26). Because prestin is in the SLC26 family, several members of which interact with CFTR, we explored the potential modulatory relationship associated with a direct, physical interaction between prestin and CFTR. Electrophysiological experiments demonstrated that cAMP-activated CFTR is capable of enhancing voltage-dependent charge displacement, a signature of OHC motility, whereas prestin does not affect the chloride conductance of CFTR.

Analysis of the oligomeric structure of the motor protein prestin.

Prestin, a member of the solute carrier family 26, is expressed in the basolateral membrane of outer hair cells. This protein provides the molecular basis for outer hair cell somatic electromotility, which is crucial for the frequency selectivity and sensitivity of mammalian hearing. It has long been known that there are abundantly expressed approximately 11-nM protein particles present in the basolateral membrane. These particles were hypothesized to be the motor proteins that drive electromotility. Because the calculated size of a prestin monomer is too small to form an approximately 11-nM particle, the possibility of prestin oligomerization was examined. We investigated possible quaternary structures of prestin by lithium dodecyl sulfate-PAGE, perfluoro-octanoate-PAGE, a membrane-based yeast two-hybrid system, and chemical cross-linking experiments. Prestin, obtained from different host or native cells, is resistant to dissociation by lithium dodecyl sulfate and behaves as a stable oligomer on lithium dodecyl sulfate-PAGE. In the membrane-based yeast two-hybrid system, homo-oligomeric interactions between prestin-bait/prestin-prey suggest that prestin molecules can associate with each other. Chemical cross-linking experiments, perfluoro-octanoate-PAGE/Western blot, and affinity purification experiments all indicate that prestin exists as a higher order oligomer, such as a tetramer, in prestin-expressing yeast, mammalian cell lines and native outer hair cells. Our data from experiments using hydrophobic and hydrophilic reducing reagents suggest that the prestin dimer is connected by a disulfide bond embedded in the prestin hydrophobic core. This stable dimer may act as the building block for producing the higher order oligomers that form the approximately 11-nM particles in the outer hair cell's basolateral membrane.

The C-terminus of prestin influences nonlinear capacitance and plasma membrane targeting.

Prestin is a unique molecular-motor protein expressed in the lateral plasma membrane of outer hair cells (OHC) in the organ of Corti of the mammalian cochlea. It is thought that prestin undergoes conformational changes driven by the cell's membrane potential. The resulting alterations in OHC-length are assumed to constitute the cochlear amplifier. Prestin is a member of the anion solute carrier family 26 (SCL26A), but it is different from other family members in its unique function of voltage-driven motility. Because the C-terminus is the least conserved region in the family, we investigated its influence with a series of deletion, point and chimeric mutants. The function and cellular expression of mutants were examined in a heterologous expression system by measurement of nonlinear capacitance (NLC) and immunofluorescence. Each mutant produced a unique mixture of patterns of cell morphologies, which were classified as to the location of prestin within the cell. The data from deletion mutants (Del516, Del525, Del630, Del590, Del709, Del719) revealed that nearly the full length (>708 amino acids) of the protein was required for normal prestin expression and function. Since most deletion mutations eliminated plasma membrane targeting, chimeric proteins were constructed by fusing prestin, at amino acid 515 or 644, with the homologous portion of the C-terminus from the two most closely related SLC26A members, pendrin and putative anion exchanger 1. These chimeric proteins were again improperly (but differently) targeted than simple truncation mutants, and all lacked functional phenotype. When two of the potential basolateral membrane-targeting motifs were mutated (Y520A/Y526A), incomplete plasma membrane expression was seen. We also show that some double point mutations (V499G/Y501H) fully express in the plasma membrane but lack NLC. These non-charged amino acids may have unrevealed important roles in prestin's function. Together, these data suggest that certain specific sequences and individual amino acids in the C-terminus are necessary for correct cellular distribution and function.

Alpha-fetoprotein triggers hepatoma cells escaping from immune surveillance through altering the expression of Fas/FasL and tumor necrosis factor related apoptosis-inducing ligand and its receptor of lymphocytes and liver cancer cells.

AIM: To investigate the mechanism of alpha-fetoprotein (AFP) in escaping from the host immune surveillance of hepatocellular carcinoma. METHODS: AFP purified from human umbilical blood was administrated into the cultured human lymphoma Jurkat T cell line or hepatoma cell line, Bel7402 in vitro. The expression of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and its receptor (TRAILR) mRNA were analyzed by Northern blot and Western blot was used to detect the expression of Fas and Fas ligand (FasL) protein. RESULTS: AFP (20 mg/L) could promote the expression of FasL and TRAIL, and inhibit the expression of Fas and TRAILR of Bel7402 cells. For Jurkat cell line, AFP could suppress the expression of FasL and TRAIL, and stimulate the expression of Fas and TRAILR. AFP also could synergize with Bel7402 cells to inhibit the expression of FasL protein and TRAIL mRNA in Jurkat cells. The monoclonal antibody against AFP (anti-AFP) could abolish these functions of AFP. CONCLUSION: AFP is able to promote the expression of FasL and TRAIL in hepatoma cells and enhance the expression of Fas and TRAILR in lymphocytes. These could elicit the escape of hepatocellular carcinoma cells from the host's lymphocytes immune surveillance.

Ca2+ signaling in HEK-293 and skeletal muscle cells expressing recombinant ryanodine receptors harboring malignant hyperthermia and central core disease mutations.

Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca(2+) release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca(2+) pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca(2+) homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca(2+) revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca(2+) transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca(2+) derangements of different degrees account for the specific cellular phenotypes of the two disorders.

Effects of cyclic nucleotides on the function of prestin.

Outer hair cells (OHCs) in the mammalian organ of Corti display electromotility, which is thought to provide the local active mechanical amplification of the cochlear response. Prestin is the key molecule responsible for OHC electromotility. Several compounds, including cGMP, have been shown to influence OHC electromotility. There are two potential cAMP/cGMP-dependent protein kinase phosphorylation sites on prestin. Whether these sites are involved in cGMP-dependent reactions is as yet unknown. In this study, prestin cDNA was transiently transfected into TSA 201 cells. Cells that expressed prestin were selected to measure non-linear capacitance (NLC), a signature of outer hair cell motility. We applied cGMP and cAMP analogues and a protein kinase G (PKG) antagonist to the cells. Furthermore, nine mutations at putative phosphorylation sites of prestin were produced. The neutral amino acid alanine replaced serine/threonine at phosphorylation sites to change the conserved phosphorylation motif in order to mimic the dephosphorylated state of prestin, whereas replacement with the negatively charged aspartic acid mimicked the phosphorylated state. The properties of such modified prestin-expressing cells were examined, through measurement of NLC and with confocal microscopy. Our data demonstrate that cGMP is significantly more influential than cAMP in modifying the non-linear, voltage-dependent charge displacement in prestin-transfected cells. The electrical properties of the single and double mutations further indicate a possible interaction between the two PKG target sites. One of these sites may influence the membrane targeting process of prestin. Finally, a new topology map of prestin is proposed.

Role of the sequence surrounding predicted transmembrane helix M4 in membrane association and function of the Ca(2+) release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor isoform 1).

The role of the sequence surrounding M4 in ryanodine receptors (RyR) in membrane association and function was investigated. This sequence contains a basic, 19-amino acid M3/M4 loop, a hydrophobic 44-49 amino acid sequence designated M4 (or M4a/M4b), and a hydrophilic M4/M5 loop. Enhanced green fluorescent protein (EGFP) was inserted into RyR1 and truncated just after the basic sequence, just after M4, within the M4/M5 loop, just before M5 and just after M5. The A52 epitope was inserted into RyR2 and truncated just after M4a. Analysis of these constructs ruled out a M3/M4 transmembrane hairpin and narrowed the region of membrane association to M4a/M4b. EGFP inserted between M4a and M4b in full-length RyR2 was altered conformationally, losing fluorescence and gaining trypsin sensitivity. Although it was accessible to an antibody from the cytosolic side, tryptic fragments were membrane-bound. The expressed protein containing EGFP retained caffeine-induced Ca(2+) release channel function. These results suggest that M4a/M4b either forms a transmembrane hairpin or associates in an unorthodox fashion with the cytosolic leaflet of the membrane, possibly involving the basic M3/M4 loop. The expression of a mutant RyR1, Delta4274-4535, deleted in the sequence surrounding both M3 and M4, restored robust, voltage-gated L-type Ca(2+) currents and Ca(2+) transients in dyspedic myotubes, demonstrating that this sequence is not required for either orthograde (DHPR activation of sarcoplasmic reticulum Ca(2+) release) or retrograde (RyR1 increase in DHPR Ca(2+) channel activity) signals of excitation-contraction coupling. Maximal amplitudes of L-currents and Ca(2+) transients with Delta4274-4535 were larger than with wild-type RyR1, and voltage-gated sarcoplasmic reticulum Ca(2+) release was more sensitive to activation by sarcolemmal voltage sensors. Thus, this region may act as a negative regulatory module that increases the energy barrier for Ca(2+) release channel opening.

Central core disease mutations R4892W, I4897T and G4898E in the ryanodine receptor isoform 1 reduce the Ca2+ sensitivity and amplitude of Ca2+-dependent Ca2+ release.

Three CCD (central core disease) mutants, R4892W (Arg4892-->), I4897T and G4898E, in the pore region of the skeletal-muscle Ca2+-release channel RyR1 (ryanodine receptor 1) were characterized using a newly developed assay that monitored Ca2+ release in the presence of Ca2+ uptake in microsomes isolated from HEK-293 cells (human embryonic kidney 293 cells), co-expressing each of the three mutants together with SERCA1a (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1a). Both Ca2+ sensitivity and peak amplitude of Ca2+ release were either absent from or sharply decreased in homotetrameric mutants. Co-expression of wild-type RyR1 with mutant RyR1 (heterotetrameric mutants) restored Ca2+ sensitivity partially, in the ratio 1:2, or fully, in the ratio 1:1. Peak amplitude was restored only partially in the ratio 1:2 or 1:1. Reduced amplitude was not correlated with maximum Ca2+ loading or the amount of expressed RyR1 protein. High-affinity [3H]ryanodine binding and caffeine-induced Ca2+ release were also absent from the three homotetrameric mutants. These results indicate that decreased Ca2+ sensitivity is one of the serious defects in these three excitation-contraction uncoupling CCD mutations. In CCD skeletal muscles, where a mixture of wild-type and mutant RyR1 is expressed, these defects are expected to decrease Ca2+-induced Ca2+ release, as well as orthograde Ca2+ release, in response to transverse tubular membrane depolarization.

N-linked glycosylation sites of the motor protein prestin: effects on membrane targeting and electrophysiological function.

Prestin is a motor protein of outer hair cells (OHC) that plays a crucial role in mammalian hearing. Prestin is a putative N-glycoprotein with three potential N-linked glycosylation sites. It is not known whether glycosylation affects the function and activity of prestin. Therefore, the effects of N-glycosylation were investigated by producing single-point (N163Q and N166Q) or double-point mutations (NN163/166QQ and NN163/166AA) at putative N-glycosylation sites. Further, treatment with tunicamycin or glycopeptidase-F was used to determine the consequences of removing N-linked glycosylation in wild-type prestin. We determined the effects of these manipulations on prestin's cell surface expression, molecular mass, glycosylation pattern, and electrophysiological properties in different cell-types. Data indicate that prestin is a glycoprotein with N-linked glycosylation sites at N163 and N166. N163 and N166 may have differential programs for synthesis and trimming of the glycans. The N166 site appears to have greater extent of glycosylation than its companion. N-linked glycosylation is not required for plasma membrane targeting of prestin. Both glycosylated and deglycosylated prestin demonstrate non-linear capacitance, a signature of prestin's motor function. Compared to glycosylated prestin, the fully de-glycosylated protein has altered electrophysiological function, with a change in membrane potential at most effective charge transfer to more depolarized values. These data suggest that glycosylation of prestin may quantitatively affect OHC electromotility.

Alpha-fetoprotein stimulated the expression of some oncogenes in human hepatocellular carcinoma Bel 7402 cells.

AIM: To investigate the molecular mechanism of alpha-fetoprotein (AFP) on regulating the proliferation of human hepatocellular carcinoma cells. METHODS: Alpha-fetoprotein purified from human umbilical blood was added to cultured human hepatocellular carcinoma Bel 7402 cells in vitro for various treatment periods. The expression of c-fos, c-jun, and N-ras mRNA involved in proliferation and differentiation of cells was analyzed by Northern blot, and the expression of mutative p53 and p21(ras) proteins was determined by Western blot. RESULTS: The results showed that AFP (20 mg/L) stimulated mRNA expression of these oncogenes in Bel 7402 cells. The expression of c-fos mRNA increased by 51.1%, 60.9%, 96.0%, and 25.5% at 2, 6, 12, and 24 h, respectively. The expression of c-jun and N-ras mRNA reached to the maximum which increased by 81.3% and 59.9% as compared with the control after 6 h and 24 h incubation with AFP, respectively. Western blot assay also demonstrated that AFP promoted the expression of mutative p53 and p21(ras) proteins, and the increased rate of those proteins was 13.0%, 39.9%, and 70.9%, as well as 35.2%, 102.6%, and 46.8% at 6, 12, and 24 h, respectively, as compared with the control. Both human serum albumin (the same dosage as AFP) and monoclonal anti-AFP antibody failed to stimulate the expression of these oncogenes, but anti-AFP antibody could block the functions of AFP. CONCLUSION: The data indicate that AFP can stimulate the expression of some oncogenes to enhance the proliferation of human hepatocellular carcinoma Bel 7402 cells.

A new cytokine: the possible effect pathway of methionine enkephalin.

AIM: To investigate experimentally the effects of methionine enkephalin on signal transduction of mouse myeloma NS-1 cells. METHODS: The antigen determinate of delta opioid receptor was designed in this lab and the polypeptide fragment of antigen determinate with 12 amino acids residues was synthesized. Monoclonal antibody against this peptide fragment was prepared. Proliferation of Mouse NS-1 cells treated with methionine enkephalin of 1 x 10(-6) mol x L(-1) was observed. The activities of protein kinase A (PKA) and protein kinase C (PKC) were measured and thereby the mechanism of effect of methionine enkephalin was postulated. RESULTS: The results demonstrated that methionine enkephalin could enhance the proliferation of NS-1 cells and the effect of methionine enkephalin could be particularly blocked by monoclonal antibody. The activity of PKA was increased in both cytosol and cell membrane. With reference to PKC, the intracellular activity of PKC in NS-1 cells was elevated at 1 x 10(-7) mol x L(-1) and then declined gradually as the concentration of methionine enkephalin was raised. The effects of methionine enkephalin might be reversed by both naloxone and monoclonal antibody. CONCLUSION: Coupled with the findings, it indicates that the signal transduction systems via PKA and PKC are involved in the effects of methionine enkephalin by binding with the traditional opioid receptors,and therefore resulting in different biological effects.

Topology of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (RyR1).

To define the topology of the skeletal muscle ryanodine receptor (RyR1), enhanced GFP (EGFP) was fused in-frame to the C terminus of RyR1, replacing a series of C-terminal deletions that started near the beginning or the end of predicted transmembrane helices M1-M10. The constructs were expressed in HEK-293 (human embryonic kidney cell line 293) or mouse embryonic fibroblast (MEF) cells, and confocal microscopy of intact and saponin-permeabilized cells was used to determine the subcellular location of the truncated fusion proteins. The fusion protein truncated after M3 exhibited uniform cytoplasmic fluorescence, which was lost after permeabilization, indicating that proposed M', M", M1, M2, and M3 sequences are not membrane-associated. The fusion protein truncated at the end of the M4-M5 loop and containing M4 was membrane-associated. All longer truncated fusion proteins were also associated with intracellular membranes. Mapping by protease digestion and extraction of isolated microsomes demonstrated that EGFP positioned after either M5, the N-terminal half of M7 (M7a), or M8 was located in the lumen, and that EGFP positioned after either M4, M6, the C-terminal half of M7 (M7b), or M10 was located in the cytoplasm. These results indicate that RyR1 contains eight transmembrane helices, organized as four hairpin loops. The first hairpin is likely to be made up of M4a-M4b. However, it could be made up from M3-M4, which might form a hairpin loop even though M3 alone is not membrane-associated. The other three hairpin loops are formed from M5-M6, M7a-M7b, and M8-M10. M9 is not a transmembrane helix, but it might form a selectivity filter between M8 and M10.

Enhancement of proliferation of HeLa cells by the alpha-fetoprotein.

A lot of investigations showed that alpha-fetoprotein(AFP) could regulate the proliferation of tumors or neoplastic cells. In order to study the enhancement of this function, AFP was extracted from human cord blood serum and was allowed to act on HeLa cell in vitro. The cellular proliferation was investigated by using the MTT assay and the [(3)H]-incorporation, and the cell cycle was analysed with flow cytometry. Radioactive immunosorbent assay was utilized to detect cAMP accumulation, protein kinase A activity and confocal microscopy was used f or scanning intracellular calcium. The expression of mutant p53 and p21(ras) protein were analyzed by Western blotting. Our results showed that AFP (>10 mg/L ) had strong effect of enhancement on the proliferation of HeLa cells; AFP (20 mg /L) could significantly increase the concentration of cAMP (300%), Ca(2+) (154.9%) and protein kinase A activity (100%). It was also shown that the expression of mutant p53 and p21(ras) protein of HeLa cells were enhanced after t he treatment with AFP (20 mg/L) for 24 hours. Compared with control, the expression of mutant p53 protein was increased by 81.1% (24 h), p21(ras) protein was increased by 96.2% (24 h) respectively; but the monoclonal antibody to AFP blocked these functions of AFP. In conclusion, AFP enhanced the proliferation of HeLa cells; AFP may regulate the growth of the cells by influencing the transmembrane signal pathway and enhancing the expression of the oncogenes.

The intracellular mechanism of alpha-fetoprotein promoting the proliferation of NIH 3T3 cells.

AIM: The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS: The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in NIH 3T3 cells. The influences of AFP on the [cAMP]i and the activities of protein kinase A (PKA) were determined. Western blot was used to detect the change of K-ras P21 protein expression. RESULTS: The proliferation of NIH 3T3 cells treated with 0-80 mg/L of AFP was significantly enhanced. The Scatchard analysis indicated that there were two classes of binding sites with KD of 2.722 x 10(-9)M (Bmax=12810 sites per cell) and 8.931 x 10(-8)M (Bmax=119700 sites per cell) respectively. In the presence of AFP (20 mg/L), the content of cAMP and activities of PKA were significantly elevated . The level of K-ras P21 protein was upregulated by AFP at the concentration of 20 mg/L. The monoclonal antibody against AFP could reverse the effects of AFP on the cAMP content, PKA activity and the expression of K-ras p21 gene. CONCLUSION: The effect of AFP on the cell proliferation was achieved by binding its receptor to trigger the signal transduction pathway of cAMP-PKA and alter the expression of K- ras p21 gene.

The promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Bel7402 cell line.

AIM: The goal of this study was to characterize the AFP receptor, its possible signal transduction pathway and its proliferative functions in human hepatoma cell line Bel 7402. METHODS: Cell proliferation enhanced by AFP was detected by MTT assay, 3H-thymidine incorporation and S-stage percentage of cell cycle analysis. With radioactive labeled 125I-AFP for receptor binding assay; cAMP accumulation, protein kinase A activity were detected by radioactive immunosorbent assay and the change of intracellular free calcium (Ca2+i) was monitored by scanning fluorescence intensity under TCS-NT confocal microscope. The expression of oncogenes N- ras, p 53, and p21( ras ) in the cultured cells in vitro were detected by Northern blotting and Western blotting respectively. RESULTS: It was demonstrated that AFP enhanced the proliferation of human hepatoma Bel 7402 cell in a dose dependent fashion as shown in MTT assay, (3)H-thymidine incorporation and S-phase percentage up to 2-fold. Two subtypes of AFP receptors were identified in the cells with Kds of 1.3 x 10(-9)mol.L(-1) and 9.9 x10(-8)mol. (-1)L respectively. Pretreatment of cells with AFP resulted in a significant increase (625%) in cAMP accumulation. The activity of protein kinase A activity were increased up to 37.5, 122.6, 73.7 and 61.2% at treatment time point 2, 6, 12 and 24 hours. The level of intracellular calcium were elevated after the treatment of alpha-fetoprotein and achieved to 204% at 4 min. The results also showed that AFP(20mg.L(-1)) could upregulate the expression of N- ras oncogenes and p 53 and p21( ras ) in Bel 7402 cells. In the later case,the alteration were 81.1%(12h) and 97.3%(12h) respectively compared with control. CONCLUSION: These results demonstrate that AFP is a potential growth factor to promote the proliferation of human hepatoma Bel 7402 cells. Its growth-regulatory effects are mediated by its specific plasma membrane receptors coupled with its transmembrane signaling transduction through the pathway of cAMP-PKA and intracellular calcium to regulate the expression of oncogenes.