P-glycoprotein

A 2015 review of polymorphisms in ABCB1 found that "the effect of ABCB1 variation on P-glycoprotein expression (messenger RNA and protein expression) and/or activity in various tissues (e.g. the liver, gut and heart) appears to be small. Although polymorphisms and haplotypes of ABCB1 have been associated with alterations in drug disposition and drug response, including adverse events with various ABCB1 substrates in different ethnic populations, the results have been majorly conflicting, with limited clinical relevance."[7]

P-gp is a 170 kDa transmembrane glycoprotein, which includes 10-15 kDa of N-terminal glycosylation. The N-terminal half of the molecule contains 6 transmembrane domains, followed by a large cytoplasmic domain with an ATP-binding site, and then a second section with 6 transmembrane domains and an ATP-binding site that shows over 65% of amino acid similarity with the first half of the polypeptide.[8] In 2009, the first structure of a mammalian P-glycoprotein was solved (3G5U).[9] The structure was derived from the mouse MDR3 gene product heterologously expressed in Pichia pastoris yeast. The structure of mouse P-gp is similar to structures of the bacterial ABC transporter MsbA (3B5W and 3B5X)[10] that adopt an inward facing conformation that is believed to be important for binding substrate along the inner leaflet of the membrane. Additional structures (3G60 and 3G61) of P-gp were also solved revealing the binding site(s) of two different cyclic peptide substrate/inhibitors. The promiscuous binding pocket of P-gp is lined with aromatic amino acid side chains.

Through Molecular Dynamic (MD) simulations, this sequence was proved to have a direct impact in the transporter's structural stability (in the nucleotide-binding domains) and defining a lower boundary for the internal drug-binding pocket.[11]

P-gp is expressed primarily in certain cell types in the liver, pancreas, kidney, colon, and jejunum.[12] P-gp is also found in brain capillary endothelial cells.[13]

Substrate enters P-gp either from an opening within the inner leaflet of the membrane or from an opening at the cytoplasmic side of the protein. ATP binds at the cytoplasmic side of the protein. Following binding of each, ATP hydrolysis shifts the substrate into a position to be excreted from the cell. Release of the phosphate (from the original ATP molecule) occurs concurrently with substrate excretion. ADP is released, and a new molecule of ATP binds to the secondary ATP-binding site. Hydrolysis and release of ADP and a phosphate molecule resets the protein, so that the process can start again.

The protein belongs to the superfamily of ATP-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and intra-cellular membranes. ABC genes are divided into seven distinct subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, White). This protein is a member of the MDR/TAP subfamily. Members of the MDR/TAP subfamily are involved in multidrug resistance. P-gp is an ATP-dependent drug efflux pump for xenobiotic compounds with broad substrate specificity. It is responsible for decreased drug accumulation in multidrug-resistant cells and often mediates the development of resistance to anticancer drugs. This protein also functions as a transporter in the blood–brain barrier. Mutations in this gene are associated with colchicine resistance and Inflammatory bowel disease 13. Alternative splicing and the use of alternative promoters results in multiple transcript variants. [14]

P-gp transports various substrates across the cell membrane including:

• Drugs such as colchicine, desloratadine,[15] tacrolimus and quinidine.
• Chemotherapeutic agents such as topoisomerase inhibitors (i.e. etoposide, doxorubicin), microtubule-targeted drugs (i.e. vinblastine), and tyrosine kinase inhibitors (i.e. gefitinib, sunitinib).
• Lipids
• Steroids
• Xenobiotics
• Peptides
• Bilirubin
• Cardiac glycosides like digoxin
• Immunosuppressive agents
• Glucocorticoids like dexamethasone
• HIV-type 1 antiretroviral therapy agents like protease inhibitors and nonnucleoside reverse transcriptase inhibitors

Its ability to transport the above substrates accounts for the many roles of P-gp including:

• Regulating the distribution and bioavailability of drugs
  • Increased intestinal expression of P-glycoprotein can reduce the absorption of drugs that are substrates for P-glycoprotein. Thus, there is a reduced bioavailability, and therapeutic plasma concentrations are not attained. On the other hand, supratherapeutic plasma concentrations and drug toxicity may result because of decreased P-glycoprotein expression
  • Active cellular transport of antineoplastics resulting in multidrug resistance to these drugs
• The removal of toxic metabolites and xenobiotics from cells into urine, bile, and the intestinal lumen
• The transport of compounds out of the brain across the blood–brain barrier
• Digoxin uptake
• Prevention of ivermectin and loperamide entry into the central nervous system
• The migration of dendritic cells
• Protection of hematopoietic stem cells from toxins.[5]

It is inhibited by many drugs, such as amiodarone, azithromycin, captopril, clarithromycin, cyclosporine, piperine, quercetin, quinidine, quinine, reserpine, ritonavir, tariquidar, and verapamil.[16]

At the transcriptional level, the expression of P-gp has been intensively studied, and numerous transcription factors and pathways are known to play roles. A variety of transcription factors, such as p53,[17] YB-1,[18] and NF-κB[19] are involved in the direct regulation of P-gp by binding to the promoter regions of the P-gp gene. Many cell signaling pathways are also involved in transcriptional regulation of P-gp. For example, the PI3K/Akt pathway[18] and the Wnt/β-catenin pathway[20] were reported to positively regulate the expression of P-gp. Mitogen-activated protein kinase (MAPK) signaling includes three pathways: the classical MAPK/ERK pathway, the p38 MAPK pathway, and the c-Jun N-terminal kinase (JNK) pathway, all of which were reported to have implications in the regulation of the expression of P-gp. Studies suggested that the MAPK/ERK pathway is involved in the positive regulation of P-gp;[21] the p38 MAPK pathway negatively regulates the expression of the P-gp gene;[22] and the JNK pathway was reported to be involved in both positive regulation and negative regulation of P-gp.[23][24]

After 2008, microRNAs (miRNAs) were identified as new players in regulating the expression of P-gp in both transcriptional and post-transcriptional levels. Some miRNAs decrease the expression of P-gp. For example, miR-200c down-regulates the expression of P-gp through the JNK signaling pathway[23] or ZEB1 and ZEB2;[25] miR-145 down-regulates the mRNA of P-gp by directly binding to the 3'-UTR of the gene of P-gp and thus suppresses the translation of P-gp.[26] Some other miRNAs increase the expression of P-gp. For example, miR-27a up-regulates P-gp expression by suppressing the Raf kinase inhibitor protein (RKIP);[27] alternatively, miR-27a can also directly bind to the promoter of the P-gp gene, which works in a similar way with the mechanism of action of transcriptional factors.[28]

The expression of P-gp is also regulated by post-translational events, such as post-transcriptional modification, degradation, and intracellular trafficking of P-gp. Pim-1 protects P-gp from ubiquitination and the following degradation in the proteasome.[29] Small GTPases Rab5 down-regulates the endocytotic trafficking of P-gp and thus increases the functional P-gp level on the cell membrane;[30] while Small GTPases Rab4 work in an opposite way: Rab4 down-regulates the exocytotic trafficking of P-gp from intracellular compartments to the cell membrane, and therefore decreases the functional P-gp level on the cell membrane.[31]

P-gp was first characterized in 1976. P-gp was shown to be responsible for conferring multidrug resistance upon mutant cultured cancer cells that had developed resistance to cytotoxic drugs.[5][45]

The structure of mouse P-gp, which has 87% sequence identity to human P-gp, was resolved by x-ray crystallography in 2009.[9] The first structure of human P-gp was solved in 2018, with the protein in its ATP-bound, outward-facing conformation. [46]

Radioactive verapamil can be used for measuring P-gp function with positron emission tomography.[47]

P-gp is also used to differentiate transitional B cells from naive B cells. Dyes such as rhodamine 123 and MitoTracker dyes from Invitrogen can be used to make this differentiation.[48]

Single Nucleotide Polymorphism are important for the differential activity of the PGP pump.

Homozygous subjects, identified with the TT genotype, are usually more able to extrude xenobiotics from the cell. A Homozygous genotype for the allele ABCB1/MDR1 is capable of a higher absorption from the blood vessels and a lower extrusion into the lumen. Xenobiotics are extruded at a lower rate with heterozygous (CT) alleles compared to homozygous ones. [50]

Wikimedia Commons has media related to P-glycoprotein.

• P-Glycoprotein at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
• ABCB1 human gene location in the UCSC Genome Browser.
• ABCB1 human gene details in the UCSC Genome Browser.

This article incorporates text from the United States National Library of Medicine, which is in the public domain.