John V. Cox, Ph.D.
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Office: (901) 448-7080
Lab: (901) 448-7071
Office: 101F M.S.B.
Lab: 117 & 119 M.S.B.
Laboratory web page
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Research Program:
Our laboratory is interested in defining how multi-membrane spanning transporters and channels are restricted to specialized membrane domains within cells. To address this question we have investigated the molecular mechanisms involved in directing the intracellular trafficking of variant chicken AE1 anion exchangers. The erythroid AE1 anion exchanger possesses dual functions. The C-terminal transmembrane domain of the AE1 anion exchanger mediates electroneutral anion exchange, while the N-terminal cytoplasmic domain of the polypeptide provides a major attachment site for the membrane cytoskeleton with the plasma membrane of erythroid cells through its association with ankyrin, protein 4.1, and protein 4.2. The critical role of these interactions in the maintenance of erythroid cell shape and stability is best illustrated by the recently described AE1 anion exchanger knockout. In addition to their severely altered morphology and fragility, erythroid cells from these animals have approximately a 50% reduction in ankyrin, and completely lack protein 4.2.
Electroneutral anion exchangers play an important role in the regulation of cellular pH and cell volume in a variety of cell types other than erythroid cells. In polarized epithelial cells, chloride-bicarbonate exchangers function coordinately with proton pumps to dispose of bicarbonate and protons generated by intracellular carbonic anhydrase. In the acid excreting a-intercalated cell of the mammalian kidney collecting duct, proton pumping is restricted to the apical membrane, while chloride-bicarbonate exchange is localized to the basolateral membrane. In contrast, chloride-bicarbonate exchange is localized to the apical membrane of the bicarbonate-secreting b-intercalated cell. Although recent studies have indicated that the basolateral chloride-bicarbonate exchanger of a-intercalated cells is encoded by the AE1 anion exchanger gene, the molecular origin of the apical anion transporter of b-intercalated cells has not yet been defined. Interestingly, exposure of a variety of mammalian species to an acid diet results in the conversion of bicarbonate-secreting b-intercalated cells to acid-secreting a-intercalated cells. It has been suggested that this plasticity in epithelial polarity is intrinsic to these epithelial cell types. However, the molecular mechanisms involved in clearing membrane transporters from one membrane domain and repopulating the opposite membrane domain are not known.
The diversity observed among AE1 anion exchangers in chickens is greater than that seen in other species. Twelve transcripts are derived from the two erythroid-specific promoters of the chicken AE1 anion exchanger gene, P1 and P2. Three additional transcripts, AE1-3, AE1-4, and AE1-5 are derived from the P3 promoter of the chicken AE1 gene. Like AE1 variants that have been characterized in other species, the polypeptides encoded by the variant chicken AE1 transcripts differ only at the N-terminus of their cytoplasmic domains.
Recent analyses in our laboratory have indicated that the alternative N-terminal cytoplasmic domains of the variant chicken kidney AE1 anion exchangers serve as signals to direct these variant transporters to opposite membrane domains in polarized kidney epithelial cells. Transfection studies have shown that the variant chicken AE1-4 anion exchanger accumulates in the basolateral membrane of polarized MDCK cells, while the AE1-3 variant, which lacks the N-terminal 63 amino acids of AE1-4, primarily accumulates in the apical membrane. The basolateral accumulation of AE1-4 is dependent upon two cytoplasmic tyrosine residues at amino acids 44 and 47 of the polypeptide. Interestingly, either of these tyrosines is sufficient to direct efficient basolateral sorting of AE1-4. However, in the absence of both tyrosine residues, AE1-4 accumulates in the apical membrane of this polarized epithelial cell type.
Kinetic studies have shown that following delivery to the cell surface newly synthesized AE1-4 is recycled to the Golgi where it acquires additional N-linked sugar modifications. Similar studies have shown that newly synthesized chicken erythroid AE1 anion exchangers also undergo recycling to the Golgi following cell surface delivery. Interestingly, the same tyrosine residues that are involved in the basolateral accumulation of AE1-4 in MDCK cells are also required for the recycling of this variant transporter from the plasma membrane to the Golgi.
Mutants of AE1-4 that are defective in Golgi recycling are unable to associate with the detergent insoluble actin cytoskeleton of MDCK cells and are rapidly turned over. These are the first results to suggest a critical role for the actin cytoskeleton in regulating the localization and stability of a membrane transporter in epithelial cells. In addition to associating with the actin cytoskeleton in MDCK cells, coimmunoprecipitation studies have indicated that AE1-4 interacts with the major epithelial ankyrin, ankyrin 3 (ank3), suggesting a role for ank3 in regulating the intracellular trafficking and/or stability of this variant transporter in epithelial cells.
Studies using AE1/Fc receptor chimeras have revealed that two independent recycling signals exist at the N-terminus of AE1-4. One of these recycling signals corresponds to the tyrosine-dependent signal mentioned above, and it is present in all of the chicken AE1 variants except AE1-3. The other cytoplasmic recycling signal resides at the N-terminus of AE1-4, and is serine-dependent. These sorting signals are the first to be characterized for a type III membrane protein, and they share features in common with the cytoplasmic sorting signals of type I membrane proteins. However, in addition to these signals, the localization and stability of AE1-4 appears to be dependent upon its ability to associate with both the actin-based cytoskeleton and the ankyrin/spectrin based peripheral membrane cytoskeleton. Association with these cytoskeletal elements is in turn dependent upon the novel Golgi recycling activity we have characterized for this electroneutral transporter.
The question we are left with at this time is how the cytoskeletal binding activities and sorting signals of AE1 variants are regulated to direct these electroneutral transporters to specific membrane domains within cells. To begin to address this issue, we are currently using transfected MDCK cells as a model system to define the vesicular sorting pathways directed by the two independent recycling signals at the N-terminus of the AE1-4 variant, as well as the structural requirements for these two sorting signals. Studies with the actin depolymerizing drug, latrunculin B, are examining the role of the actin cytoskeleton in directing 1) the cell surface delivery; 2) recycling from the plasma membrane to the Golgi; and 3) the acquisition of detergent insolubility by AE1-4 in MDCK cells. In addition, mutagenesis studies are defining the specific sequences of AE1-4 that are necessary for its association with epithelial ank3. Having defined these sequences we will assess the role of ank3 in directing the intracellular trafficking of AE1 anion exchangers in epithelial cells. It has been proposed that association with the peripheral membrane cytoskeleton through interaction with ankyrin provides a mechanism for restricting the distribution of membrane transporters and channels, including the Na+,K+-ATPase, the voltage-dependent Na+ channel, and the amiloride-sensitive Na+ channel, to specialized membrane domains within cells. Our studies will reveal how cytoplasmic sorting signals direct the cytoskeletal association of variant AE1 anion exchangers, and the consequences of these interactions on protein localization and stability. In addition, these studies will ultimately lead to an understanding of the mechanisms that regulate the conversion of bicarbonate-secreting b-intercalated cells to acid-secreting a-intercalated cells that can be induced in vivo.
Selected Publications
Cox, K.H., and Cox, J.V. 1995. Variant chicken AE1 anion exchangers possess divergent N-terminal cytoplasmic domains. Amer. J. Phys. 268:F503-F513.
Cox, K.H., Adair-Kirk, T.L., and Cox, J.V. 1995. Four variant chicken erythroid AE1 anion exchangers: Potential role of the alternative N-terminal sequences in intracellular targeting. J. Biol. Chem. 270:19752-19760.
Cox, K.H., Adair-Kirk, T.L., and Cox, J.V. 1996. Variant AE2 anion exchanger transcripts accumulate in multiple cell types in the chicken gastric epithelium. J. Biol. Chem. 271:8895-8902.
Cox, K.H., Adair-Kirk, T.L., and Cox, J.V. 1996. Variant chicken kidney AE1 anion exchanger transcripts are derived from a single promoter by alternative splicing. Gene 173:221-226.
Ghosh, S., Cox, K.H., and Cox, J.V. 1999. Chicken erythroid AE1 anion exchangers associate with the cytoskeleton during recycling to the Golgi. Mol. Biol. Cell 10:455-469.
Adair-Kirk, T.L., Cox, K.H., and Cox, J.V. 1999. Intracellular trafficking of variant chicken kidney AE1 anion exchangers: Role of alternative N-termini in polarized sorting and Golgi recycling. Submitted.
Ghosh, S., and Cox, J.V. 1999. Role of phosphorylation in regulating the dynamics of ankyrin/AE1 anion exchanger complexes in chicken erythroid cells. Submitted.
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