Multidrug resistance-associated protein (MRP) mediated transport of daunomycin and leukotriene C4 (LTC4) in isolated plasma membrane vesicles.

Studies have been carried out to examine in vitro drug transport in plasma membrane vesicles isolated from HL60/ADR cells that overexpress MRP. The results demonstrate that glutathione (GSH) enhances transport of daunomycin. A greater increase in transport activity occurs when the reaction is carried out in the presence of both GSH and sodium chloride. Sodium chloride alone has no effect on daunomycin transport. It has also been observed that GSH in the presence of sodium chloride induces a major increase in the transport level of LTC4. Thus far, no metal ion other than sodium chloride has been found to be active in the drug transport system. Kinetic analysis reveals that GSH in the presence of sodium chloride greatly reduces Km and increases Vmax, for daunomycin. Additional studies show that ATPase activity in isolated plasma membrane from HL60/ADR cells is greatly enhanced in the presence of both GSH and sodium chloride. These results suggest the possibility that GSH and sodium chloride stimulate MRP-mediated transport as a result of increased ATPase activity.

Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells.

Two membrane glycoproteins acting as energy-dependent efflux pumps, mdr-encoded P-glycoprotein (P-gp) and the more recently described multidrug resistance-associated protein (MRP), are known to confer cellular resistance to many cytotoxic hydrophobic drugs. In the brain, P-gp has been shown to be expressed specifically in the capillary endothelial cells forming the blood-brain barrier, but localization of MRP has not been well characterized yet. Using RT-PCR and immunoblot analysis, we have compared the expression of P-gp and Mrp1 in homogenates, isolated capillaries, primary cultured endothelial cells, and RBE4 immortalized endothelial cells from rat brain. Whereas the mdr1a P-gp-encoding mRNA was specifically detected in brain microvessels and mdr1b mRNA in brain parenchyma, mrp1 mRNA was present both in microvessels and in parenchyma. However, Mrp1 was weakly expressed in microvessels. Mrp1 expression was higher in brain parenchyma, as well as in primary cultured brain endothelial cells and in immortalized RBE4 cells. This Mrp1 overexpression in cultured brain endothelial cells was less pronounced when the cells were cocultured with astrocytes. A low Mrp activity could be demonstrated in the endothelial cell primary monocultures, because the intracellular [3H]vincristine accumulation was increased by several MRP modulators. No Mrp activity was found in the cocultures or in the RBE4 cells. We suggest that in rat brain, Mrp1, unlike P-gp, is not predominantly expressed in the blood-brain barrier endothelial cells and that Mrp1 and the mdr1b P-gp isoform may be present in other cerebral cells.

Mutagenesis of the putative nucleotide-binding domains of the multidrug resistance associated protein (MRP). Analysis of the effect of these mutations on MRP mediated drug resistance and transport.

Studies were conducted to examine the functional role of the nucleotide-binding domains of MRP in drug resistance and drug transport in isolated membrane vesicles. In vivo studies were conducted by preparing stable transfectants of HeLa cells with wild-type MRP cDNA or MRP cDNAs which had been mutated at certain nucleotide binding domains (NBD). Stable transfectants producing equivalent amounts of the MRP encoded protein P190 were used in this study. The results demonstrated that deletions in the C-motif of NBD1 or the A-motif of NBD2 have a pronounced effect in reducing resistance levels to chemotherapeutic agents. Certain single-site mutations in lysines in these same motifs also reduce IC(50) values. It has also been observed that mutation of the MRP NBDs results in an increase in drug accumulation and a reduction in drug efflux. Additional studies have been carried out in which recombinant baculovirus containing either wild-type MRP or MRP containing mutated NBDs was prepared and used to infect SF21 insect cells. Using this system we have analyzed the effects of these mutations on in vitro transport of leukotriene C(4) (LTC(4)) 17 beta-estradiol 17 (beta-D-glucuronide)(E(2)17betaG) and daunomycin in membrane vesicles prepared from baculovirus infected cells. The results demonstrate that deletions and site-specific mutations in MRP NBDs greatly reduce the ATP dependent transport of all three substrates. The results of these studies conducted both in vivo and in vitro demonstrate that the NBDs of MRP function in a cooperative manner and are critical for the transport activity of the MRP encoded protein P190. These studies also identify specific lysines in NBD1 and NBD2 which are important for optimal MRP activity.

Functional analysis of the nucleotide binding domains of the multidrug resistance protein (MRP).

HeLa cells were transfected with full-length multidrug resistance protein (MRP) cDNA and with MRP cDNAs that had been mutated at certain nucleotide binding domains. Stable transfectants were isolated and those producing equivalent amounts of P190 were tested in cytotoxicity assays using a variety of chemotherapeutic agents. The results demonstrate that deletions in the C-motif of NBD1 or the A-motif of NBD2 have a pronounced effect in reducing resistance levels to adriamycin, vincristine, or etoposide (VP-16). Single-site mutations of lysine in these same motifs reduce IC50 values but less than that observed with the deletion mutants. Additional studies have demonstrated an increase in drug accumulation and reduction in drug efflux in NBD deletion and single-site mutants. The results of this study therefore identify two lysines of the NBD A- and C-motifs that are critical for MRP-mediated multidrug resistance. The results also provide definitive evidence that resistance occurring as a result of MRP overexpression is related to enhanced levels of an ATP-dependent efflux pump.

Evidence that SP1 modulates transcriptional activity of the multidrug resistance-associated protein gene.

In previous studies, we have cloned and sequenced a 5'-end region of the multidrug resistance-associated protein (MRP) gene that contains promoter activity as assessed through transient transfections of constructs contained in a pCAT basic reporter plasmid. In the present study, using a series of deletion mutants, evidence was obtained that the SP1 binding sites contained in the promoter are essential for optimal MRP transcriptional activity. These results were supported by the finding that introduction of site-specific mutations into the wild-type SP1 sequence produced a major reduction in CAT activity. DNase I protection assays also demonstrated that SP1 sites are protected from hydrolysis with proteins from nuclei of a variety of cell lines. Gel mobility-shift assays with proteins extracted from CHO, HeLa, HL60, or HL60/ADR demonstrated the presence of a protein that bound to the wild-type SP1 sequence but not to an SP1 sequence containing site-specific mutations. The mobility shift with nuclear extracts was closely similar to that occurring after incubating purified SP1 protein with wild-type SP1 sequence. DNA supershift experiments with antibody to SP1 strongly suggest that the complexes formed with nuclear extracts contain the SP1 protein.

Expression and characterization of the multidrug resistance-associated protein in insect cells infected with a recombinant baculovirus.

The protein encoded by the multidrug resistance-associated protein (MRP) gene was examined after infection of SF21 insect cells with recombinant baculovirus containing a full-length MRP cDNA. The time course of appearance of the protein as determined by western blot analysis revealed that maximum levels occurred 2 days postinfection. The amount of MRP made in this system was somewhat variable, but levels that were about 4-fold greater than that found in HL60/ADR cells could be achieved. The protein appeared to be full-length but was present in a highly deglycosylated form. The P170 (MRP) was phosphorylated and located exclusively in membranes of infected cells. P170 (MRP) synthesized in this system was capable of carrying out the ATP-dependent transport of leukotriene C4 into isolated membrane vesicles. The results thus indicate that MRP synthesized in insect cells is functional and has properties similar to the authentic protein found overexpressed in certain multidrug-resistant isolates.

Phosphorylation of the multidrug resistance associated protein gene encoded protein P190.

Recent studies suggest that multidrug resistance of HL60/ADR cells is related to an overexpression of the MRP (multidrug resistance associated protein) gene which encodes a 190-kDa ATP-binding membrane glycoprotein. In the present study we have further characterized P190 and have examined phosphorylation properties of the protein. The results demonstrate that P190 is highly phosphorylated and that the phosphate groups are metabolically active and undergo cycles of phosphorylation and dephosphorylation in the cell. Serine is the single amino acid phosphorylated in P190 and the phosphate groups are contained in nine tryptic peptides. Experiments have also been conducted to analyze the effect of various protein kinase inhibitors on phosphorylation levels of P190. The results show that H-7, staurosporine, and chelerythrine can reduce the phosphorylation of this protein. In the presence of both H-7 (200 microM) and staurosporine (200 nM) the phosphorylation of P190 is completely blocked. It has also been found that in the presence of these agents there is a major increase in drug accumulation and concomitant inhibition in drug efflux of resistant cells. These results therefore suggest the possibility that certain phosphate groups of protein P190 play an important role in modulating drug accumulation in resistant cells.

Cloning and sequence analysis of the promoter region of the MRP gene of HL60 cells isolated for resistance to adriamycin.

Non-P-glycoprotein multidrug resistance of HL60/ADR cells appears to be related to overexpression of the MRP gene. Recent studies suggest that this gene may play an important role in a new form of cell resistance to certain chemotherapeutic agents. To examine mechanisms regulating transcriptional activity of this gene, a 2.2-kilobase 5'-flanking sequence of MRP has been isolated from a genomic library prepared from HL60/ADR cells. The 2.2-kilobase DNA fragment linked to the chloramphenicol acetyltransferase (CAT) gene in a reporter plasmid was found to be capable of driving expression of this gene in transient transfection experiments. This DNA containing promoter activity has been sequenced in its entirety and found to contain multiple putative regulatory sites. A series of deletion mutants linked to the CAT reporter gene was used to examine functional domains of the 2.2-kilobase sequence. The results suggest that promoter activity is contained in nucleotides -91 to +103 in a GC-rich region of the MRP genome. Promoter activity contained within this sequence, however, is modulated by both positive and negative regulatory elements. Certain of the regulatory sites contain consensus sequences for positive and negative regulatory elements which have been found in the promoter regions of other genes. Primer extension analysis indicates the presence of multiple major transcriptional start sites from the MRP promoter. Sequence analysis of MRP genomic and complementary DNAs has defined the exon/intron boundaries and the organization of a portion of the 5'-end region of the MRP genome. The results of these studies thus provide new insight in site-specific domains which may function in the regulation of MRP gene expression.

Analysis of MRP gene expression and function in HL60 cells isolated for resistance to adriamycin.

In an effort to define clearly the basis of non-P-glycoprotein multidrug resistance in HL60/ADR cells, we have analyzed expression of MRP mRNA levels and the MRP-encoded protein in resistant cells and also in resistant cells that have undergone a reversion to drug sensitivity. The results demonstrate that an MRP cDNA containing 5'-end coding sequences reacts with a 6-kb RNA, which is overexpressed in the resistant isolate. As resistant cells revert to drug sensitivity there is essentially a complete loss of the 6-kb RNA. Southern blot analysis indicates that the MRP gene is amplified compared to the copy number found in sensitive cells. Revertant cells no longer contain amplified MRP sequences. Western blot analysis has been conducted using an antibody prepared against the carboxyl terminus (15 amino acids) of the deduced sequence of the MRP-encoded protein. The antibody is reactive with a 190-kDa protein (P190) and with two closely migrating proteins of 65 and 70 kDa (P70), which are overexpressed in plasma membranes and endoplasmic reticulum of resistant cells. Both proteins are greatly reduced in revertant cells. Growth of cells in the presence of tunicamycin demonstrates that both P190 and P70 are glycosylated, with the deglycosylated forms migrating in polyacrylamide gels as proteins of 165 kDa and 45 kDa, respectively. Additional antisera have also been prepared against sequence domains contained in the C-terminal region of P190. These antisera are reactive with both P190 and P70. Antisera directed against sequences of the amino terminal region of P190 do not react with P70.(ABSTRACT TRUNCATED AT 250 WORDS)

The MRP gene associated with a non-P-glycoprotein multidrug resistance encodes a 190-kDa membrane bound glycoprotein.

HL60 cells isolated for resistance to Adriamycin (HL60/ADR) overexpress a 190-kDa ATP binding protein which has a minor sequence homology with P-glycoprotein. It has also been observed that HL60/ADR overexpress the MRP gene which was first identified as a component of a non-P-glycoprotein mediated multidrug resistance of H69/ADR cells [Cole et al., Science (Washington DC), 258: 1650, 1992]. A complementary DNA of MRP has been cloned and based on the deduced sequence encodes a member of the superfamily of proteins which bind ATP and function in various transport processes [Cole et al., Science (Washington DC), 258: 1650, 1992]. In view of this it was of interest to identify the protein encoded by MRP and determine if it may be related to p190. In the present study we have prepared antisera against three synthetic peptides which correspond to the deduced sequence of the MRP protein. Proteins reactive with the antisera have been examined in HL60/ADR cells using Western blot analysis. All antisera react with a 190 kDa protein contained in membranes of resistant but not sensitive cells. One antiserum used for further studies is not reactive with P-glycoprotein contained in membranes of HL60 cells isolated for resistance to vincristine. Analysis of subcellular fractions demonstrates that p190 is present primarily in the endoplasmic reticulum with lower levels also present in plasma membranes. Treatment of HL60/ADR cells with tunicamycin results in the appearance of a 165-kDa resistance associated protein which reacts with the antipeptide serum. The results of this study therefore demonstrate that the MRP gene encodes a 190-kDa membrane bound glycoprotein.

Non-P-glycoprotein multidrug resistance in cell lines which are defective in the cellular accumulation of drug.

Non-Pgp mdr related to a defect in drug accumulation has now been documented in a number of different cell lines exposed to certain cytotoxic agents. In studies conducted thus far most isolates have been obtained after selection in either adriamycin or mitoxantrone. The work in this area is in its early stages and very little is known about the molecular events which contribute to this mode of drug resistance. At the present time no protein with drug binding properties comparable to Pgp has been identified in non-Pgp mdr isolates. Evidence based on the finding that all isolates do not respond in the same way to reversal agents such as verapamil suggests the possibility that more than one mechanism may exist for non-Pgp mdr. Future studies may thus reveal that cells contain a multiplicity of genes which upon transcriptional activation can function to alter drug transport processes and thus contribute to the development of mdr. Identifying and characterizing these genes will be important since they may function in transport systems of normal cells. The exact identify of proteins which contribute to non-Pgp mdr remains to be determined. One protein designated P190 has been found to be overexpressed in cell lines of human promyelocytic leukemia, lung and adenocarcinoma treated with adriamycin. The protein also is increased in some clinical samples from patients undergoing chemotherapy. P190 which has a minor sequence homology with Pgp can bind ATP and may thus contribute to the energy dependent drug efflux systems found in cells containing this protein. Transfection studies with a P190 cDNA should determine whether this protein actually contributes to drug resistance. Many other protein changes have been detected in non-Pgp mdr cells but the importance of these in resistance also remains to be determined. In some systems a particular protein change can be identified in multiple independent isolates suggesting a correlation between the development of resistance and the presence of this cellular alteration. Experiments conducted thus far on the mechanism of non-Pgp mdr are intriguing. Studies utilizing fluorescence microscopy to follow the fate of daunomycin suggests that the drug passes to the interior of the cell and eventually localizes in the Golgi apparatus. Drug located at this site may move directly into an efflux pathway for rapid extrusion from the cell. Evidence also indicates that as drug leaves the Golgi some may be sequestered into other organelles such as lysosomes or mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemosensitisation and drug accumulation effects of cyclosporin A, PSC-833 and verapamil in human MDR large cell lung cancer cells expressing a 190k membrane protein distinct from P-glycoprotein.

The doxorubicin-selected multidrug resistant (MDR) human large cell lung cancer line COR-L23/R, lacks P-glycoprotein but shows a drug accumulation deficit. It does however overexpress a 190k membrane protein which shares an epitope with, but is otherwise distinct from, P-glycoprotein. The resistant cells show only a small sensitisation to vincristine and daunorubicin on treatment with cyclosporin A and its more potent analogue, PSC-833 despite an increase in drug accumulation. Verapamil, another effective resistance modifier in P-glycoprotein MDR cells, is slightly more effective. Fluorescent daunorubicin distributes in the cytoplasm and nucleus of sensitive parent COR-L23 cells but is confined to cytoplasmic perinuclear vesicles in resistant cells. Addition of cyclosporin A or PSC-833 slightly increases cytoplasmic fluorescence whereas verapamil also increases nuclear fluorescence. Resistance in this non-P-glycoprotein MDR line, COR-L23/R where these resistance modifiers have little effect may be associated with expression of the 190k protein.

Drug transport mechanisms in HL60 cells isolated for resistance to adriamycin: evidence for nuclear drug accumulation and redistribution in resistant cells.

HL60 cells isolated for resistance to Adriamycin are multidrug resistant and defective in the cellular accumulation of drug. These cells do not contain detectable levels of P-glycoprotein. At the present time the mechanism by which HL60/Adr cells reduce drug levels is not known. To gain insight into the molecular basis of this system we have analyzed transport pathways and the distribution of daunomycin in drug-resistant HL60 cells. Using a cell fractionation technique we find that the major portion of daunomycin accumulates in the nucleus of both sensitive and resistant cells. Further studies reveal, however, that under efflux conditions drug is retained in the nuclei of sensitive cells but rapidly removed from the nuclei of the resistant isolate. Essentially identical results are obtained when daunomycin distribution and transport are analyzed by fluorescence microscopy. A number of agents which alter transport processes have been tested for their effect on drug accumulation in resistant cells. Thus we find that brefeldin A, which disassembles Golgi, and various lysosomotropic agents such as chloroquine and methylamine do not affect drug levels. In contrast the protonophores nigericin and monensin induce an increase in drug accumulation and inhibit efflux. The results of this study thus suggest that resistance in HL60/Adr cells is related to a mechanism whereby drug is transported to the nucleus and thereafter rapidly redistributed to the extracellular space. The molecular basis of this transport pathway is not known.

Regulation of ribosomal protein S25 in HL60 cells isolated for resistance to adriamycin.

The ribosomal protein S25 gene is highly overexpressed in HL60 cells isolated for resistance to adriamycin. In contrast there is no overexpression of 3 other ribosomal genes which code for proteins S14, S17 and S24. Studies with an antibody against a synthetic peptide of the S25 protein show that although the S25 gene is overexpressed in resistant cells there is no corresponding increase in the levels of S25 protein. These results suggest that the r-protein levels are highly regulated by translational controls or protein turnover.

Detection and characterization of membrane protein changes in multidrug resistant HL-60 cells.

Antisera were prepared against fractionated membrane proteins of HL-60 cells isolated for resistance to adriamycin. Analysis of these antisera revealed that one (GSBl) was capable of detecting major protein changes in three independent isolates selected for anthracycline resistance. Thus, in studies using western blot analysis, the antiserum was found to be reactive with two proteins of 130 and 150 kDa which are present in plasma membranes of resistant but not sensitive cells. The antibody also reacted with a plasma membrane protein of 180 kDa that is present in sensitive cells but is increased in resistant isolates. Additional studies showed that P180 was greatly increased in both plasma membranes and endoplasmic reticulum in sensitive cells induced to differentiate in the presence of 12-O-tetradecanoylphorbol13-acetate (TPA). Resistant cells treated under identical conditions showed only a slight increase in the levels of P180. TPA had no effect on the levels of P150 or P130. In contrast, differentiation of HL-60 cells in the presence of dimethylsulfoxide (DMSO) resulted in the induction of P150 expression with major levels of protein contained in plasma membranes. DMSO has essentially no effect on the levels of plasma membrane P180, P150, or P130 in HL-60/Adr cells. These results therefore demonstrate a strong correlation between the development of resistance and the overexpression of proteins reactive with the GSBl antiserum. The results also show that development of anthracycline resistance in HL-60 cells results in the overexpression of P150, a protein associated with the differentiation of myeloid cells to granulocytes.

HL-60 cells isolated for resistance to vincristine are defective in 12-O-tetradecanoylphorbol-13-acetate induced differentiation and the formation of a functional AP-1 complex.

HL-60 cells isolated for resistance to vincristine are multidrug resistant and defective in the cellular accumulation of drug. Further studies demonstrate that these cells are also highly defective in 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation to macrophages. Analysis of this system demonstrates that certain protooncogenes which may contribute to differentiation are expressed at similar levels in sensitive and resistant cells. Thus, treatment of cells with TPA results in a reduction in the levels of c-myb and c-myc mRNA, while the expression of c-fos, c-jun, and junB is greatly enhanced. Immunoprecipitation experiments also demonstrate a TPA induced increase in the c-jun protein in both sensitive and resistant cells. Gel mobility shift assays show that TPA induces AP-1 formation in sensitive cells, whereas in parallel experiments with the HL-60/Vinc isolate, AP-1 is essentially absent. It has been found, however, that in resistant cells which have reverted to drug sensitivity, the levels of TPA inducible AP-1 is essentially identical to that of sensitive cells. Revertant and sensitive cells differentiate at similar levels in the presence of TPA. These studies therefore demonstrate that HL-60/Vinc cells are defective in the TPA induction of a functional AP-1 complex and that this may account for the inability of these cells to differentiate to macrophages. The molecular basis of the finding that AP-1 is not formed in resistant cells remains to be determined.

The gene encoding vacuolar H(+)-ATPase subunit C is overexpressed in multidrug-resistant HL60 cells.

Previous studies have suggested that vacuolar H(+)-ATPase activity may play a role in modulating drug transport mechanism in multidrug resistant HL60 cells. In the present study we have used a cDNA of human vacuolar H(+)-ATPase subunit C (SC-H(+)-ATPase) to analyze expression of this gene in HL60 cells isolated for resistance to adriamycin or vincristine. The results demonstrate that development of resistance to either agent results in a major increase in the levels of SC-H(+)-ATPase mRNA. Furthermore in resistant cells which have partially reverted to drug sensitivity there is a parallel reduction in SC-H(+)-ATPase mRNA levels. Southern blot analysis shows that the SC-H(+)-ATPase gene is not amplified in the resistant cells. These results therefore demonstrate a correlation between the development of multidrug resistance and enhanced expression of the SC-H(+)-ATPase gene.

Cloning and sequencing a cDNA encoding human ribosomal protein S25.

A full-length cDNA clone has been isolated from a cDNA library prepared from mRNA of adriamycin-resistant human leukemia HL60 cells. The nucleotide sequence of this cDNA has been determined and the protein coded for by the gene identified. The cDNA encodes a polypeptide of 125 amino acids (aa) with a deduced Mr of 13750. The deduced aa sequence of this protein has 56% homology to yeast ribosomal protein S31. Western-blot analysis using antibodies directed against a synthetic peptide based on the deduced aa sequence identifies the gene product as the human ribosomal protein S25.

Involvement of vacuolar H(+)-adenosine triphosphatase activity in multidrug resistance in HL60 cells.

HL60 cells isolated for resistance to vincristine (HL60/Vinc cells) or doxorubicin (HL60/Adr cells) contain enhanced levels of an energy-dependent drug efflux pump. HL60/Vinc cells contain the drug transporter P-glycoprotein, whereas the HL60/Adr isolate does not. In the present study, we examined the possible involvement of vacuolar H(+)-adenosine triphosphatase (H(+)-ATPase) activity in drug resistance in HL60 cells. We utilized bafilomycin A1, an agent which selectively inhibits vacuolar H(+)-ATPase activity at low concentrations. The results showed that bafilomycin A1 induced a major increase in drug accumulation and inhibited drug efflux in both HL60/Adr cells and HL60/Vinc cells. Similar results were obtained with 7-chloro-4-nitrobenz-2-oxa 1,3 diazole, an agent which is also capable of inhibiting vacuolar H(+)-ATPase. Azide, an inhibitor of F1F0 mitochondrial ATPase, and vanadate and ouabain, which are inhibitors of E1E2-type ATPase, did not affect drug levels in resistant cells. We also observed that bafilomycin A1 did not compete with [3H]azidopine binding to P-glycoprotein. Thus, bafilomycin A1 does not appear to function as a substrate for P-glycoprotein. These results suggest an involvement of vacuolar H(+)-ATPase activity in the pathway of drug efflux from HL60/Adr cells and HL60/Vinc cells. The mechanism of this action remains to be determined.

Analysis of P-glycoprotein phosphorylation in HL60 cells isolated for resistance to vincristine.

In the present study we have analyzed the involvement of phosphorylation in the function of P-glycoprotein and have also examined sites of phosphorylation along the P-glycoprotein polypeptide chain. The results show that in HL60 cells isolated for resistance to vincristine the protein kinase inhibitor staurosporine induces a major inhibition in the phosphorylation of P-glycoprotein. Further studies show that under the same conditions in which staurosporine inhibits P-glycoprotein phosphorylation there is a concomitant increase in cellular drug accumulation and a major inhibition in drug efflux. Additional studies using pulse-chase experiments show that the P-glycoprotein phosphate groups are metabolically active and that the protein undergoes rapid cycles of phosphorylation and dephosphorylation in the cell. Structural analyses demonstrate that cleavage of 32P-labeled P-glycoprotein at Asp-Pro linkages with formic acid results in the formation of a major phosphorylated peptide of 35 kDa and a minor peptide of 42 kDa. Western blot analysis using site-specific anti-sera against P-glycoprotein suggests that P35 represents a phosphorylated fragment containing P-glycoprotein amino acids 446-744. Analysis of tryptic peptides using site-specific antisera identifies a second major phosphorylated region of P-glycoprotein which contains amino acids 745-1088. These studies thus suggest that phosphorylation plays an important role in the biological activity of P-glycoprotein. The results also indicate that two adjacent internal regions are highly phosphorylated in the P-glycoprotein molecule.