Mu Protein Research Articles

Repeat Organization and Epigenetic Regulation of the DH-Cμ Domain of the Immunoglobulin Heavy-Chain Gene Locus

The first steps of murine immunoglobulin heavy-chain (IgH) gene recombination take place within a chromosomal domain that contains diversity (D(H)) and joining (J(H)) gene segments, but not variable (V(H)) gene segments. Here we show that the chromatin state of this domain is markedly heterogeneous. Specifically, only 5'- and 3'-most D(H) gene segments carry active chromatin modifications, whereas intervening D(H)s are associated with heterochromatic marks that are maintained by ongoing histone deacetylation. The intervening D(H)s form part of a tandemly repeated sequence that expresses tissue-specific, antisense oriented transcripts. We propose that the intervening D(H) genes are actively suppressed by repeat-induced epigenetic silencing, which is reflected in their infrequent representation in DJ(H) junctions compared to the flanking D(H) genes.

Precursor of human adenovirus core polypeptide Mu targets the nucleolus and modulates the expression of E2 proteins.

We have examined the subcellular localization properties of human adenovirus 2 (HAdV-2) preMu and mature Mu (pX) proteins as fusions with enhanced green fluorescence protein (EGFP). We determined that preMu is exclusively a nucleolar protein with a single nucleolar accumulation signal within the Mu sequence. In addition, we noted that both preMu-EGFP and Mu-EGFP are excluded from adenovirus DNA-binding protein (DBP)-rich replication centres in adenovirus-infected cells. Surprisingly, we observed that cells in which preMu-EGFP (but not Mu-EGFP) is transiently expressed prior to or shortly after infection with Ad2 did not express late adenovirus genes. Further investigation suggested this might be due to a failure to express pre-terminal protein (preTP) from the E2 region, despite expression of another E2 protein, DBP. Deletion mutagenesis identified a highly conserved region in the C terminus of preMu responsible for these observations. Thus our data suggest that preMu may play a role in modulating accumulation of proteins from the E2 region.

MACROARRAY ANALYSIS REVEALS A STRAIN-INDUCED OXIDANT RESPONSE IN PULMONARY EPITHELIAL CELLS

Mechanical strain initiates a variety of responses in pulmonary epithelial cells. The signaling pathways and molecular alterations leading to these responses remain unclear. To identify novel signal transduction pathways activated by strain, macroarray analysis was performed on strained pulmonary epithelial cells. Glutathione S-transferase (GST) pi, GST mu, and heat shock protein (HSP)-27 were increased by strain. Western blotting verified that increases in cDNA of these redox-related molecules resulted in an increase in protein. Phosphorylation of HSP-27 was increased after strain, further supporting the role of HSP-27 in strain-induced signal transduction. Strain-induced oxidative stress was verified with the oxidant-sensitive dye dichlorodihydrofluorescein diacetate.

Abundant expression of mu and delta opioid receptor mRNA and protein in the cerebellum of the fetal, neonatal, and adult rat

Opioid receptor proteins and mRNAs have been localized to a variety of regions within the rat brain. It is generally accepted that within the lobes of the rat cerebellum, only delta opioid receptor (DOR) is expressed. This is in contrast to that observed in humans and rabbits which express both mu opioid receptor (MOR) and DOR. In this study, we report detection of MOR as well as DOR protein by immunohistochemical localization, and mRNA by fluorescent in situ hybridization (FISH) within Purkinje cells (PK) and the granular layer of neonatal (P6) and adult rat cerebellum. Expression of MOR mRNA was also detected within cells of the molecular layer, but at lower levels than those seen within the PK cells. Abundant expression of MOR and DOR mRNA was detected in the external germinal layer of the immature cerebellum of the fetal (E16) rat, supporting a role for MOR and DOR in regulating neurogenesis of the cerebellum. In addition, using exon-specific cRNA probes, exons 1 and 4, which are both found in the MOR-1 splice variant mRNA, were detected in PK cells in the cerebellum and also within deep cerebellar nuclei in the adult.

Ontogeny and subcellular localization of rat liver mitochondrial branched chain amino-acid aminotransferase.

Branched chain amino-acid aminotransferase (BCAT) activity is present in fetal liver but the developmental pattern of mitochondrial BCAT (BCATm) expression in rat liver has not been studied. The aim of this study was to determine the activity, protein and mRNA concentration of BCATm in fetal and postnatal rat liver, and to localize this enzyme at the cellular and subcellular levels at both developmental stages. Maximal BCAT activity and BCATm mRNA expression occurred at 17 days' gestation in fetal rat liver and then declined significantly immediately after birth. This pattern was observed only in liver; rat heart showed a different developmental pattern. Fetal liver showed intense immunostaining to BCATm in the nuclei and mitochondria of hepatic cells and blood cell precursors; in contrast, adult liver showed mild immunoreactivity located only in the mitochondria of hepatocytes. BCAT activity in isolated fetal liver nuclei was 0.64 mU x mg(-1) protein whereas it was undetectable in adult liver nuclei. By Western blot analysis the BCATm antibody recognized a 41-kDa protein in fetal liver nuclei, and proteins of 41 and 43 kDa in fetal liver supernatant. In adult rat liver supernatant, the BCATm antibody recognized only a 43-kDa protein; however, neither protein was detected in adult rat liver nuclei. The appearance of the 41-kDa protein was associated with the presence of the highly active form of BCATm. These results suggest the existence of active and inactive forms of BCAT in rat liver.

Immunoglobulin heavy chain expression shapes the B cell receptor repertoire in human B cell development.

Developing B cells must pass a series of checkpoints that are regulated by membrane-bound Ig(mu) through the Igalpha-Igbeta signal transducers. To determine how Ig(mu) expression affects B cell development and Ab selection in humans we analyzed Ig gene rearrangements in pro-B cells from two patients who are unable to produce Ig(mu) proteins. We find that Ig(mu) expression does not affect V(H), D, or J(H) segment usage and is not required for human Igkappa and Iglambda recombination or expression. However, the heavy and light chains found in pro-B cells differed from those in peripheral B cells in that they showed unusually long CDR3s. In addition, the Igkappa repertoire in Ig(mu)-deficient pro-B cells was skewed to downstream Jkappas and upstream Vkappas, consistent with persistent secondary V(D)J rearrangements. Thus, Ig(mu) expression is not required for secondary V(D)J recombination in pro-B cells. However, B cell receptor expression shapes the Ab repertoire in humans and is essential for selection against Ab's with long CDR3s.

The PTPμ Protein-tyrosine Phosphatase Binds and Recruits the Scaffolding Protein RACK1 to Cell-Cell Contacts

PTPmu, an Ig superfamily receptor protein-tyrosine phosphatase, promotes cell-cell adhesion and interacts with the cadherin-catenin complex. The signaling pathway downstream of PTPmu is unknown; therefore, we used a yeast two-hybrid screen to identify additional PTPmu interacting proteins. The membrane-proximal catalytic domain of PTPmu was used as bait. Sequencing of two positive clones identified the scaffolding protein RACK1 (receptor for activated protein C kinase) as a PTPmu interacting protein. We demonstrate that RACK1 interacts with PTPmu when co-expressed in a recombinant baculovirus expression system. RACK1 is known to bind to the src protein-tyrosine kinase. This study demonstrates that PTPmu association with RACK1 is disrupted by the presence of constituitively active src. RACK1 is thought to be a scaffolding protein that recruits proteins to the plasma membrane via an unknown mechanism. We have shown that the association of endogenous PTPmu and RACK1 in a lung cell line is increased at high cell density. We also demonstrate that the recruitment of RACK1 to both the plasma membrane and cell-cell contact sites is dependent upon the presence of the PTP mu protein in these cells. Therefore, PTPmu may be one of the proteins that recruits RACK1 to points of cell-cell contact, which may be important for PTPmu-dependent signaling in response to cell-cell adhesion.

Morphine Upregulates mu Opioid Receptors of Human and Monkey Lymphocytes

Opioid receptors of subtypes delta, kappa, and mu similar to those found in brain cells have been identified in immune cells. The current study demonstrates by competitive polymerase chain reaction the treatment of human lymphocytic cells with morphine resulting in an increased amount of gene expression of mu opioid receptors. Antibodies against the MOR-1, the neuronal mu opioid receptor, were used in Western blot analysis of mu proteins and the results revealed a single band of ∼50 kDa, the intensity of which was increased by morphine treatment. Similar results of mu opioid receptor activation were observed when monkey lymphocytes were treated with morphine. These studies suggest that in addition to causing an immune effect through communication with the neuroendocrine system, the psychoactive drug morphine may modulate immune functions by acting directly on the mu opioid receptors expressed on lymphocytes.

Towards integrating vectors for gene therapy: expression of functional bacteriophage MuA and MuB proteins in mammalian cells.

Bacteriophage Mu has one of the best studied, most efficient and largest transposition machineries of the prokaryotic world. To harness this attractive integration machinery for use in mammalian cells, we cloned the coding sequences of the phage factors MuA and MuB in a eukaryotic expression cassette and fused them to a FLAG epitope and a SV40-derived nuclear localization signal. We demonstrate that these N-terminal extensions were sufficient to target the Mu proteins to the nucleus, while their function in Escherichia coli was not impeded. In vivo transposition in mammalian cells was analysed by co-transfection of the MuA and MuB expression vectors with a donor construct, which contained a miniMu transposon carrying a Hygromycin-resistance marker (Hyg(R)). In all co-transfections, a significant but moderate (up to 2.7-fold) increase in Hyg(R) colonies was obtained if compared with control experiments in which the MuA vector was omitted. To study whether the increased efficiency was the result of bona fide Mu transposition, integrated vector copies were cloned from 43 monoclonal and one polyclonal cell lines. However, in none of these clones, the junction between the vector and the chromosomal DNA was localized precisely at the border of the Att sites. From our data we conclude that expression of MuA and MuB increases the integration of miniMu vectors in mammalian cells, but that this increase is not the result of bona fide Mu-induced transposition.

Development of Phase II Xenobiotic Metabolizing Enzymes in Differentiating Murine Clara Cells

Glutathione S-transferases (GSTs) and epoxide hydrolases (EHs) protect cells from exogenous insult by detoxifying electrophilic compounds. Little is known about these enzyme systems during postnatal lung development. This study was designed to help establish whether the heightened neonatal susceptibility of the lung to bioactivated cytotoxicants is the result of inadequate ability to detoxify reactive intermediates. We compared the distribution of immunoreactive protein and enzymatic activity of GSTs and EHs in isolated distal airways during pre- and postnatal development in lungs of mice from 16 days gestation to 9 weeks postnatal age (adult). GST alpha, mu, and pi class protein expression in fetal and postnatal lung varied by isozyme and age. Isozymes alpha and mu are expressed at low levels before birth, high levels on postnatal day 7, low levels between postnatal days 14 and 21, high levels at postnatal day 28, and slightly lower levels in adults. Immunoreactive protein of isozyme pi has a peak expression on gestational day 18 and again on postnatal day 4, is undetectable at postnatal day 21, and is at peak levels in the adult mouse lung. GST activity in distal airways increased with age. Microsomal EH protein expression increased in intensity with age, while activity was similar in airways from all ages. We conclude that in the mouse lung (1) cellular expression of glutathione S-transferase varies by age and isozyme and does not increase with increasing age, (2) airway glutathione S-transferase activity increases with increasing age and does not correlate with immunoreactive protein expression, and (3) airway microsomal epoxide hydrolase activity does not increase, even though immunoreactive protein expression does increase with age.

Targeting p53, hdm2, and CD19: vaccination and immunologic strategies.

Peptides presented by class I major histocompatibility complex (MHC) molecules and derived from normal self-proteins that are expressed at elevated levels by cells from a variety of human (Hu) malignancies provide, in theory, potential target antigens for a broad-spectrum, cytotoxic T lymphocyte (CTL)-based immunotherapy of cancer and hematologic malignancies. However, as such tumor- and leukemia-associated self-proteins are also expressed at low levels in some types of normal tissues, such as thymus, spleen and lymphohemopoietic cells, these self-MHC-self-peptide complexes may also represent thymic and/or peripheral tolerogens, thereby preventing immune responses. This is particularly true for class I MHC-peptide complexes expressed by bone marrow-derived cells in the thymus, as such expression would cause negative selection of immature thymic T cells with high avidity for self-MHC-self-peptide complexes. This intrathymic deletion of potentially self-reactive T cells could result in a peripheral T cell repertoire purged of CTL precursors with sufficient avidity to recognize natural tumor associated self-epitopes presented by class I MHC molecules on tumor cells. HLA-transgenic (Tg) mice provide the basis of an experimental strategy that exploits species differences between Hu and murine (Mu) protein sequences in order to circumvent self-tolerance and obtain HLA-restricted CTL specific for epitopes derived from tumor- and leukemia-associated Hu self proteins, such as p53, Her-2/neu, hdm2 and CD19.

Development of a PCR test based on a gene region associated with the pathogenicity of Pasteurella multocida serotype B:2, the causal agent of Haemorrhagic Septicaemia in Asia

We have developed a PCR assay to detect Pasteurella multocida serotype B:2, the causal agent of Haemorrhagic Septicaemia (HS) in Asia. Nucleotide sequence determination of a 16S rRNA–23S rRNA PCR product unique to B:2 strains was shown to share amino acid sequence homology with a bacteriophage Mu protein. Primers designed from this sequence when tested against a panel of isolates recovered from a wide geographical area and representing a large range of bacterial genera and species, were found to specifically amplify DNA from P. multocida, serotype B:2. Southern hybridisation confirmed the presence of this sequence in only the B:2 serotype of P. multocida, suggesting an association between bacterial virulence and the presence of bacteriophage genes in the bacterial genome. The results of this study demonstrate the potential application of PCR to the diagnosis of HS in cattle and buffalo in Asia. Application of PCR to support diagnosis of HS will greatly improve accuracy, laboratory response time, and will facilitate rational deployment of resources for controlling this disease.

A truncated heavy chain protein relieves the requirement for surrogate light chains in early B cell development.

Early B cell development depends upon the surface expression of Ig heavy chain protein (mu) in a signaling complex known as the pre-B cell receptor (pre-BCR). In addition to mu, the pre-BCR consists of the surrogate light chains VpreB and lambda5 and the transmembrane signal transduction proteins Ig-alpha and Ig-beta. Expression of this complex is associated with changes in surface marker expression, gene transcription, and Ig gene rearrangement. Mutations preventing the expression of either mu or lambda5 result in developmental arrest, but the precise roles of the various components of the pre-BCR remain unclear. Using mice transgenic for a surface-expressed, but truncated, form of mu that cannot associate with surrogate light chains, we have studied the role of surrogate light chains in B cell development. We found that expression of the truncated mu transgene resulted in changes in surface marker expression, germline kappa locus transcription, and V(D)J recombinase targeting indistinguishable from those induced by intact mu protein. These experiments lead us to conclude that surrogate light chains, while necessary for the assembly of the wild-type pre-BCR, are not directly involved in pre-BCR signaling or otherwise required for early B cell development.

Inducible expression of N-methyl- d-aspartate receptor, and delta and mu opioid receptor messenger RNAs and protein in the NT2-N human cell line

Retinoic acid treatment of NT-era2/cl.D1 (NT2) cells, a human teratocarcinoma cell line, yields 95% pure cultures of terminally differentiated neuronal cells. Concomitant with their terminal differentiation into neurons, NT2 cells are induced by retinoic acid to express neuronal N-methyl- d-aspartate receptor channels, which are fully functional. We determined the effects of retinoic acid-induced differentiation of NT2 cells on the levels of N-methyl- d-aspartate, delta opioid and mu opioid receptor messenger RNAs. RNA levels were measured using quantitative solution hybridization assays. The riboprobes were complementary to major portions of the coding regions of the N-methyl- d-aspartate, delta opioid and mu opioid receptor complementary DNAs. After four weeks of exposure to 10 μM retinoic acid, followed by four weeks of treatment with mitotic inhibitors (1 μM of cytosine arabinoside, 10 μM of fluorodeoxyuridine and 10 μM of uridine) the levels of N-methyl- d-aspartate receptor messenger RNA in differentiated NT2-N cells increased 10-fold, delta opioid receptor messenger RNA increased three-fold, and mu opioid receptor messenger RNA increased four-fold. Northern blot analysis revealed two transcripts for the N-methyl- d-aspartate receptor messenger RNA (4.2 and 4.4 kb) and two transcripts for delta opioid receptor messenger RNA (7.0 and 11.0 kb). To determine whether the increases in messenger RNAs were accompanied by an increased synthesis of the respective proteins, we examined the immunoperoxidase localization of N-methyl- d-aspartate receptor and delta opioid receptor antisera. N-Methyl- d-aspartate receptor-like immunoreactivity was seen within the cell bodies as well as on the processes of the retinoic acid-differentiated cells. Although delta opioid receptor-like immunoreactivity was detected within the soma of isolated cells prior to retinoic acid treatment, the apparent number of these labelled cells and their ramified processes were markedly enhanced following retinoic acid differentiation. These results demonstrate parallels between the inducible expression of the N-methyl- d-aspartate and opioid receptor messenger RNAs and proteins during the acquisition of the fully differentiated neuronal phenotype in cultured NT2 cells. Retinoic acid-differentiated NT2 cells express increased levels for the N-methyl- d-aspartate, delta opioid and mu opioid receptor messenger RNAs, providing the opportunity to study the interactions among these receptor systems in human terminally differentiated neuronal cells in culture.

Down-regulation of terminal deoxynucleotidyl transferase by Ig heavy chain in B lineage cells.

The enzyme terminal deoxynucleotidyl transferase (TdT) adds nontemplate-derived nucleotides (N regions) to the junctions between recombining variable, diversity, and joining segments of Ig genes. The relative paucity of N regions in Ig light chains, together with the down-regulation of TdT transcription in pre-B cells (prior to light chain production), suggested that production of IgM heavy chain (mu) protein might negatively regulate TdT expression. In this study, we examined the effect of mu production on TdT gene expression in B lineage subsets from normal mice, from recombination-deficient mice (SCID and Rag-1-) carrying mu transgenes, and in transformed pro-B cell lines transfected with mu constructs. In normal mice, TdT is sharply down-regulated at the early pre-B stage in which cells have just completed productive mu rearrangement. Furthermore, the expression of mu transgenes in pro-B stage cells from recombination-deficient mice results in a similar decrease. Finally, transfection of genomic constructs encoding mu into pro-B cell lines results in a marked reduction of TdT expression. Taken together, these findings indicate that mu protein production results in the down-regulation of TdT. The ability of mu transgenes to alter TdT expression in cell lines also suggests that signaling through the pre-B receptor does not necessarily require interaction with an external stromal cell-derived ligand.

Neuronal Nitric-oxide Synthase-μ, an Alternatively Spliced Isoform Expressed in Differentiated Skeletal Muscle

Nitric oxide (NO) functions as a molecular mediator in numerous processes in cellular development and physiology. Differential expression and regulation of a family of three NO synthase (NOS) gene products help achieve this diversity of action. Previous studies identify post-translational modification and interaction of NOS with specific protein targets as tissue-specific modes of regulation. Here, we show that alternative splicing specifically regulates neuronal NOS (nNOS, type I) in striated muscle. nNOS in skeletal muscle is slightly more massive than nNOS from brain owing to a 102-base pair (34-amino acid) alternatively spliced segment between exons 16 and 17. Following purification, this novel nNOS mu isoform has similar catalytic activity to that of nNOS expressed in cerebellum. nNOS mu appears to function exclusively in differentiated muscle as its expression occurs coincidentally with myotube fusion in culture. An isoform-specific antibody detects nNOS mu protein only in skeletal muscle and heart. This study identifies alternative splicing as a means for tissue-specific regulation of nNOS and reports the first additional protein sequence for a mammalian NOS since the original cloning of the gene family.

Regulatory factors acting at the bacteriophage Mu middle promoter.

Lytic development of bacteriophage Mu proceeds through three phases of transcription: early, middle, and late. Initiation of middle transcription from Pm requires the phage-encoded activator, Mor. An examination of the sequences surrounding the promoter revealed possible binding sites for Mu proteins A and c, as well as for Escherichia coli integration host factor. Promoter fragments containing 5' and 3' deletions were fused to the lacZ reporter gene and assayed for activity after induction of a Mu prophage or a plasmid-borne mor gene. Sequences upstream of position -62 and downstream of +10 were dispensable for promoter activity. In DNase I footprinting with both crude extract and purified protein, Mor protected Pm sequences from position -56 to -33. Mutations disrupting the dyad symmetry of the terminator of early transcription overlapping the Mor binding site did not reduce promoter activity, suggesting that the symmetry per se is not required for Mor binding or Pm activation. Purified Mu lysogenic repressor (c) also bound to Pm, overlapping the Mor binding site. Production of large amounts of repressor in vivo reduced Mor-dependent promoter activity nearly 10-fold. Promoters with mutations in the repressor binding site showed a reduction in this repressor-mediated inhibition of Pm activity.

Characterization of activators and inhibitors of protein kinase C mu.

In order to investigate regulatory mechanisms and to identify potential substrates of a novel member of the protein kinase C (PKC) family, PKC mu, specific antibodies have been raised against unique amino- and carboxy-terminal regions. PKC mu kinase activity was studied upon immunoprecipitation from stably transfected cell lines as well as from the A549 carcinoma cell line expressing the endogenous PKC mu gene. Cell fractionation revealed that PKC mu is predominantly found in the particulate fraction, suggesting an association with the membrane or membrane-bound structures. In vitro kinase assays with immunoprecipitated PKC mu demonstrated a Ca2+ independent enhancement of constitutive autophosphorylation activity by phosphatidylserine. Despite a limited in vitro phorbol ester response, an apparent phorbol ester activation of PKC mu was observed when cell cultures, instead of immunoprecipitated enzyme, were treated with either phorbol 12-myristate 13-acetate or 1,2 dioleoyl-sn-glycerol. Both in vitro autophosphorylation and substrate phosphorylation of myelin basic protein and histone III were enhanced under these conditions. However, long-term treatment with the phorbol ester did not result in downregulation of PKC mu protein levels and kinase activity. Studies with several protein kinase inhibitors revealed a novel sensitivity profile of PKC mu, with no inhibition by calphostin C, reduced sensitivity to staurosporine but, compared to other PKCs, an approximately 60-fold higher sensitivity to the selective PKA inhibitor H89. Together, the data presented here show that localization of PKC mu and regulation of its kinase activity differ from that of other PKCs suggesting a novel function of PKC mu in intracellular signal pathways.

Ig heavy chain protein controls B cell development by regulating germ-line transcription and retargeting V(D)J recombination.

The membrane-associated form of Ig heavy chain (mu) protein has been implicated as a critical regulator of B cell development. Mutant mice unable to produce the membrane form of mu protein fail to produce mature B cells. Splenic B cells from mice transgenic for a functionally rearranged Ig mu gene show a marked decrease in endogenous heavy chain gene rearrangement. We have analyzed the effects of a human mu transgene on the regulation of V(D)J recombination during B cell development in the mouse fetal liver. We found that mu transgenic and wild-type littermate mice begin kappa light chain gene rearrangement at the same time during development but the transgenic mice show a striking increase in the frequency of kappa gene rearrangement. The transgenic mice also show an increase in the levels of a germ-line kappa gene transcript known to be associated with kappa gene rearrangement. D-to-JH heavy chain gene rearrangement is unaffected throughout development by the presence of the mu transgene. Endogenous heavy chain gene V-to-DJH rearrangement occurs with similar frequency in transgenic and nontransgenic fetal livers during midgestation but is reduced in late gestation mu transgenic fetal liver. We show that this decrease in rearrangement is associated with a decrease in unrearranged VH gene transcription. Furthermore, we show that changes in the frequencies of rearranged kappa and mu genes are accompanied by changes in the frequencies of dsDNA breaks, a V(D)J recombination reaction intermediate associated with each of these loci. We propose that membrane-associated mu protein regulates B cell development by signaling a change in the pattern of unrearranged Ig gene transcriptional activity, thereby retargeting the V(D)J recombinase.

Transfection of mu MDR 1 inhibits Na(+)-independent Cl-/-HCO3 exchange in Chinese hamster ovary cells.

We have used single-cell photometry to measure intracellular pH (pHi) for several MDR cell lines constructed by stably transfecting LR73 chinese hamster ovary fibroblasts with mutant and wild type murine MDR 1 genes. In addition, plasma membrane electrical potential (delta psi) has been measured for the same cells by the K+/valinomycin null point titration method using the ratiometric styryl probe di-4-ANEPPS. Both the untransfected, parental cell line and a cell line expressing substantial mutant MDR 1 protein (K432R/K1074R) that is unable to confer the MDR phenotype are found to have delta psi > or = -40 (+/- 5) mV and pHi < or = 7.16 (+/- 0.03) units. In contrast, MDR cell lines constructed by transfecting wild type mu MDR 1 cDNA are found to exhibit delta psi from 15 to 19 mV lower and pHi from 0.13 to 0.34 units higher. A cell line that overexpresses crippled MDR protein (S941F) that is not resistant to colchicine or doxorubicin, but which is resistant to vinblastine [Gros, P., Dhir, R., Croop, J., & Talbot, F. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7289-7293], exhibits elevated pHi and slightly elevated delta psi, relative to LR73. Northern and western blot analyses confirm the substantial overexpression of the mu MDR genes and proteins in these lines, as well as the mild overexpression of endogenous hamster p-GP mRNA in some lines. In general agreement with previous studies that examined myeloma cells overexpressing hu MDR 1 protein [Roepe, P.D., Wei, L.-Y., Cruz, J., & Carlson, D. (1993) Biochemistry 32, 11042-11056] we find that overexpression of wild type mu MDR 1 protein inhibits Cl(-)- and -HCO3-dependent pHi homeostasis. Via single-cell photometry studies we now conclude that this is due to inhibition of Na(+)-independent Cl-/-HCO3 exchange (strict anion exchange or AE). As concluded previously for other MDR cells, decreased AE activity is not due to decreased expression of the exchanger; in fact, again similar to previous work [Roepe et al. (1993) Biochemistry 32, 11042-11056], we find increased levels of AE mRNA in some MDR cell lines. Models that may explain these data that are also consistent with the known physiology of cells that endogenously express MDR protein are suggested. These data are consistent with a model for MDR protein function wherein overexpression of the protein decreases delta psi and/or elevates pHi via Cl(-)- and -HCO3-dependent mechanisms.