Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510
megalin; epithelia; polarity; sorting; signal; apical
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ARTICLE |
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WHENEVER A CELL employs the sequences encoded within its genome to direct the synthesis of a new polypeptide, it solves a fundamental problem in information processing. Decades of experiments have defined many of the mechanisms through which cells translate their genetic information into structural and enzymatic activities. Translation, however, is only the first (and perhaps best understood) of several issues in information processing confronted by a cell engaged in protein synthesis. In subsequent steps, information presumably embedded within the newly synthesized protein itself is used to guide numerous aspects of the protein's posttranslational processing. One critically important posttranslational processing event results in the targeting of the protein to its site of ultimate functional residence. The remarkable anisotropy of eukaryotic cells could not be maintained if every newly synthesized polypeptide were free to distribute itself randomly throughout the cytoplasm. Instead, machinery must exist that can use the information inherent in each protein to mediate its appropriate delivery.
The problem of polypeptide sorting is especially well illustrated by the membrane proteins of polarized epithelial cells. The plasma membranes of such cells (including those that line the renal tubules or the lumen of the gut) are divided into two distinct domains that manifest markedly different protein compositions (5). This compositional asymmetry is responsible for many of the important physiological properties of epithelia, including the renal reabsorption of fluid and electrolytes and the intestinal absorption of nutrients. Clearly, polarized epithelial cells must be able to distinguish among newly synthesized plasmalemmal proteins to effect their sorting to the appropriate cell surface domain. Furthermore, the cells must possess mechanisms that are capable of retaining these proteins in their respective domains following their arrival at the cell surface (3). Much remains to be learned about the nature of the sorting information that specifies each protein's subcellular localization and about the cellular sorting apparatus that interprets and acts upon this information.
Newly synthesized membrane proteins can follow a variety of pathways en route to achieving a polarized distribution in epithelial cells (19). They can travel from the secretory pathway directly to their appropriate and final destination (14, 23, 25). For this vectorial delivery to occur, apical and basolateral membrane proteins must be segregated from one another and sorted into distinct carrier vesicles before their arrival at the cell surface (16). Alternatively, apical and basolateral proteins can share a carrier vesicle to one or the other surface domain. Those that find themselves in the wrong place are then re-internalized into vesicular carriers that transcytose them to their proper locale (1, 26, 27, 35). Finally, membrane proteins can be randomly delivered to the entire plasmalemma and subsequently stabilized and retained only in select locations (37). In each of these cases, the participating apical and basolateral proteins must be endowed with sorting signals that actively specify their respective fates.
Several classes of signals that mediate basolateral targeting have been
identified. Perhaps the best studied of these are short
tyrosine-containing sequences of the form NPXY or YXX, where X can
be any amino acid and
is a hydrophobic residue (20, 21). These motifs are displayed on the cytoplasmic surfaces of
membrane proteins and appear to function through interactions with
components of clathrin adaptor complexes (11, 12).
Dileucine motifs have also been shown to direct basolateral targeting
and probably do so through similar types of interactions (22,
32).
Much less is known about the signals that drive apical sorting. Proteins that exhibit the capacity to partition into glycosphingolipid-rich membrane domains appear to be apically sorted by virtue of this behavior (4, 36). The nature or degree of a membrane protein's glycosylation may also, at least in some cases, render it a substrate for apical delivery (2, 18). Only a few specific protein sequences that function as apical sorting signals have been elucidated. The cytoplasmic COOH-terminal tail of rhodopsin contains a sequence that appears to function as an autonomous apical sorting signal when this protein is expressed in epithelial cells (7). The extreme COOH termini of CFTR, the sodium/phosphate cotransporter (NaPi II), and the GABA transporter GAT-3 appear to direct apical localization through interactions with PDZ domain-containing proteins (10, 13, 24, 28-30). In the case of the gastric H,K-ATPase, apical distribution is specified, at least in part, by sequences that appear to reside in and flank a transmembrane domain (9).
The current article in focus by Takeda et al. (Ref. 38; see C1105-C1113 in this issue) provides evidence for a new and potentially quite interesting addition to the short list of apical targeting sequences. In a series of elegant and quantitative experiments, these authors identify a region within the cytoplasmic COOH-terminal tail of megalin that appears to be required for this protein's apical sorting in renal epithelial cells. Megalin spans the membrane once, and its cytoplasmic COOH terminus possesses a wealth of sequences that resemble motifs involved in sorting or in various types of protein-protein interactions (15, 34). Takeda et al. carried out a deletion analysis to define a stretch of 30 amino acids toward the center of the 213-residue tail sequence that appears to be necessary to direct apical targeting when the protein is expressed by transfection in MDCK cells.
Megalin is thought to function physiologically primarily as an endocytic receptor, and it is a substrate for rapid internalization from the plasma membrane (6). Many proteins that are subject to endocytosis possess cytoplasmic tyrosine-containing motifs that are related but not identical to basolateral sorting signals (39). These motifs direct the parent protein's interactions with the AP-2 clathrin adaptor endocytic machinery (31) and are absolutely required for efficient internalization. The cytoplasmic tail of megalin possesses three such candidate motifs, one of which resides in the domain defined by Takeda et al. (38) to play a role in apical sorting. Ligand uptake studies demonstrate that the tyrosine motif embedded within the apical sorting segment does not play a role in endocytosis, whereas both of the others do contribute to this process. Targeting experiments indicate that megalin is delivered directly to the MDCK cell apical membrane. Together, these observations suggest that megalin's apical sorting motif specifies the protein's initial targeting rather than subsequent phenomena such as postdelivery stabilization or post-endocytic redistribution.
Not surprisingly, the identification of this novel apical sorting signal raises at least as many questions as it answers. Perhaps the most pressing among these relates to the involvement of this sequence's tyrosine residue in apical targeting. As noted earlier, tyrosine-based motifs have been found to confer basolateral sorting on a large number of proteins in a variety of epithelial cell types (20, 22). Mutagenesis studies will be required to determine whether the tyrosine residue is, in fact, a critical component of the apical sorting motif. Should this prove to be the case, our current understanding of the mechanisms through which sorting signals are interpreted may need to be broadened. Recent data suggest that tyrosine-based basolateral sorting signals are recognized by the µ1B subunit of the AP-1 adaptor, either during the protein's initial passage through the Golgi complex or at the level of the recycling endosome following the protein's internalization from the plasmalemma (11, 12). In either case, this association appears to be important for the proper steady-state localization of at least some basolateral proteins endowed with tyrosine signals. If a tyrosine-based signal can be shown to specify apical sorting, then it will be fascinating to determine whether this motif is interpreted through interactions with components of clathrin adaptor complexes and, if so, to establish the identity of adaptor subunits involved. Are there as-yet unidentified adaptor subunits that function to recognize tyrosine-based apical signals? Alternatively, could µ1B serve double duty, acting as an engine of basolateral or apical sorting, depending on a given tyrosine signal's precise molecular context? If the megalin apical signal proves to be dependent on a tyrosine, these questions are likely to become the focus of intense investigation.
Takeda et al. (38) have very convincingly demonstrated
that the 30-amino acid segment they identified is necessary to ensure megalin's apical accumulation. It remains to be determined, however, whether this signal is, of itself, sufficient to account for this behavior. It is possible that the apical motif constitutes a portion of
a more extensive signal domain whose composition includes additional residues that are either adjacent or noncontiguous in the protein's linear sequence. Evidence suggesting that an apical sorting determinant can be created from noncontiguous protein domains comes from studies of
the gastric H,K-ATPase -subunit. The sequences that flank this
protein's fourth transmembrane segment can collaborate to form a
functional apical sorting signal (9). One widely used technique for establishing whether a sorting motif functions
autonomously is to create a protein construct in which this motif is
appended to a polypeptide that contains no other dominant sorting
information (8). By determining whether the megalin motif
can redirect an "unsorted" construct to the apical surface in
transfected epithelial cells, it should be possible to assess whether
the information it contains is both necessary and sufficient to mediate
its parent protein's targeting.
The language of sorting signals is not universal. It appears that at least in several cases the same signal can be differentially interpreted by different epithelial cell types (17, 33). It will be interesting to determine whether the megalin apical sorting motif is able to specify this destination in epithelial cells that manifest distinct morphologies and are specialized to perform distinct functions. Finally, it will be fascinating to identify the components of the cellular sorting machinery that interact with and interpret the megalin signal. These components may include adaptor subunits or some other known or novel constituents of the cell's trafficking apparatus. Elucidating these protein partners will provide new and important mechanistic insights into the processes that epithelial cells use to generate their remarkable asymmetry. Takeda et al. (38) have presented an exciting new observation whose further pursuit may reveal new perspectives on the manifold processes of cell sorting.
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ACKNOWLEDGEMENTS |
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I thank all of the members of my laboratory group for helpful discussions and critical comments.
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FOOTNOTES |
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Work cited from my laboratory was supported by National Institutes of Health Grants GM-42136 and DK-17433.
Address for reprint requests and other correspondence: M. J. Caplan, Dept. of Cellular and Molecular Physiology, Yale Univ. School of Medicine, 333 Cedar St., New Haven, CT 06510 (E-mail: michael.caplan{at}yale.edu).
10.1152/ajpcell.00004.2003
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REFERENCES |
---|
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---|
1.
Bartles, JR,
Feracci HM,
Stieger B,
and
Hubbard AL.
Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation.
J Cell Biol
105:
1241-1251,
1987[Abstract].
2.
Benting, JH,
Rietveld AG,
and
Simons K.
N-glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in Madin-Darby canine kidney cells.
J Cell Biol
146:
313-320,
1999
3.
Brown, D,
and
Stow JL.
Protein trafficking and polarity in kidney epithelium: from cell biology to physiology.
Physiol Rev
76:
245-297,
1996
4.
Brown, DA,
and
Rose JK.
Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.
Cell
68:
533-544,
1992[ISI][Medline].
5.
Caplan, MJ.
Membrane polarity in epithelial cells: protein sorting and establishment of polarized domains.
Am J Physiol Renal Physiol
272:
F425-F429,
1997
6.
Christensen, EI,
and
Birn H.
Megalin and cubilin: multifunctional endocytic receptors.
Nat Rev Mol Cell Biol
3:
256-266,
2002[Medline].
7.
Chuang, JZ,
and
Sung CH.
The cytoplasmic tail of rhodopsin acts as a novel apical sorting signal in polarized MDCK cells.
J Cell Biol
142:
1245-1256,
1998
8.
Deen, PM,
Van Balkom BW,
Savelkoul PJ,
Kamsteeg EJ,
Van Raak M,
Jennings ML,
Muth TR,
Rajendran V,
and
Caplan MJ.
Aquaporin-2: COOH terminus is necessary but not sufficient for routing to the apical membrane.
Am J Physiol Renal Physiol
282:
F330-F340,
2002
9.
Dunbar, LA,
Aronson P,
and
Caplan MJ.
A transmembrane segment determines the steady-state localization of an ion-transporting adenosine triphosphatase.
J Cell Biol
148:
769-778,
2000
10.
Egan, ME,
Glockner-Pagel J,
Ambrose C,
Cahill PA,
Pappoe L,
Balamuth N,
Cho E,
Canny S,
Wagner CA,
Geibel J,
and
Caplan MJ.
Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells.
Nat Med
8:
485-492,
2002[ISI][Medline].
11.
Folsch, H,
Ohno H,
Bonifacino JS,
and
Mellman I.
A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells.
Cell
99:
189-198,
1999[ISI][Medline].
12.
Gan, Y,
McGraw TE,
and
Rodriguez-Boulan E.
The epithelial-specific adaptor AP1B mediates post-endocytic recycling to the basolateral membrane.
Nat Cell Biol
4:
605-609,
2002[ISI][Medline].
13.
Gisler, SM,
Stagljar I,
Traebert M,
Bacic D,
Biber J,
and
Murer H.
Interaction of the type IIa Na/Pi cotransporter with PDZ proteins.
J Biol Chem
276:
9206-9213,
2001
14.
Gottardi CJ and Caplan MJ. Delivery of
Na+,K+-ATPase in polarized epithelial cells.
Science 260: 552-554, discussion 554-556, 1993.
15.
Gotthardt, M,
Trommsdorff M,
Nevitt MF,
Shelton J,
Richardson JA,
Stockinger W,
Nimpf J,
and
Herz J.
Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction.
J Biol Chem
275:
25616-25624,
2000
16.
Griffiths, G,
and
Simons K.
The trans Golgi network: sorting at the exit site of the Golgi complex.
Science
234:
438-443,
1986[ISI][Medline].
17.
Gu, HH,
Ahn J,
Caplan MJ,
Blakely RD,
Levey AI,
and
Rudnick G.
Cell-specific sorting of biogenic amine transporters expressed in epithelial cells.
J Biol Chem
271:
18100-18106,
1996
18.
Gut, A,
Kappeler F,
Hyka N,
Balda MS,
Hauri HP,
and
Matter K.
Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins.
EMBO J
17:
1919-1929,
1998
19.
Le Gall, AH,
Yeaman C,
Muesch A,
and
Rodriguez-Boulan E.
Epithelial cell polarity: new perspectives.
Semin Nephrol
15:
272-284,
1995[ISI][Medline].
20.
Matter, K.
Epithelial polarity: sorting out the sorters.
Curr Biol
10:
R39-R42,
2000[ISI][Medline].
21.
Matter, K,
Hunziker W,
and
Mellman I.
Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants.
Cell
71:
741-753,
1992[ISI][Medline].
22.
Matter, K,
Yamamoto EM,
and
Mellman I.
Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells.
J Cell Biol
126:
991-1004,
1994[Abstract].
23.
Mays, RW,
Siemers KA,
Fritz BA,
Lowe AW,
van Meer G,
and
Nelson WJ.
Hierarchy of mechanisms involved in generating Na/K-ATPase polarity in MDCK epithelial cells.
J Cell Biol
130:
1105-1115,
1995[Abstract].
24.
Milewski, MI,
Mickle JE,
Forrest JK,
Stafford DM,
Moyer BD,
Cheng J,
Guggino WB,
Stanton BA,
and
Cutting GR.
A PDZ-binding motif is essential but not sufficient to localize the C terminus of CFTR to the apical membrane.
J Cell Sci
114:
719-726,
2001
25.
Misek, DE,
Bard E,
and
Rodriguez-Boulan E.
Biogenesis of epithelial cell polarity: intracellular sorting and vectorial exocytosis of an apical plasma membrane glycoprotein.
Cell
39:
537-546,
1984[ISI][Medline].
26.
Mostov, KE,
and
Deitcher DL.
Polymeric immunoglobulin receptor expressed in MDCK cells transcytoses IgA.
Cell
46:
613-621,
1986[ISI][Medline].
27.
Mostov, KE,
Verges M,
and
Altschuler Y.
Membrane traffic in polarized epithelial cells.
Curr Opin Cell Biol
12:
483-490,
2000[ISI][Medline].
28.
Moyer, BD,
Denton J,
Karlson KH,
Reynolds D,
Wang S,
Mickle JE,
Milewski M,
Cutting GR,
Guggino WB,
Li M,
and
Stanton BA.
A PDZ-interacting domain in CFTR is an apical membrane polarization signal.
J Clin Invest
104:
1353-1361,
1999
29.
Moyer, BD,
Duhaime M,
Shaw C,
Denton J,
Reynolds D,
Karlson KH,
Pfeiffer J,
Wang S,
Mickle JE,
Milewski M,
Cutting GR,
Guggino WB,
Li M,
and
Stanton BA.
The PDZ-interacting domain of cystic fibrosis transmembrane conductance regulator is required for functional expression in the apical plasma membrane.
J Biol Chem
275:
27069-27074,
2000
30.
Muth, TR,
Ahn J,
and
Caplan MJ.
Identification of sorting determinants in the C-terminal cytoplasmic tails of the gamma-aminobutyric acid transporters GAT-2 and GAT-3.
J Biol Chem
273:
25616-25627,
1998
31.
Ohno, H,
Stewart J,
Fournier MC,
Bosshart H,
Rhee I,
Miyatake S,
Saito T,
Gallusser A,
Kirchhausen T,
and
Bonifacino JS.
Interaction of tyrosine-based sorting signals with clathrin-associated proteins.
Science
269:
1872-1875,
1995[ISI][Medline].
32.
Rapoport, I,
Chen YC,
Cupers P,
Shoelson SE,
and
Kirchhausen T.
Dileucine-based sorting signals bind to the beta chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif- binding site.
EMBO J
17:
2148-2155,
1998
33.
Roush, DL,
Gottardi CJ,
Naim HY,
Roth MG,
and
Caplan MJ.
Tyrosine-based membrane protein sorting signals are differentially interpreted by polarized Madin-Darby canine kidney and LLC-PK1 epithelial cells.
J Biol Chem
273:
26862-26869,
1998
34.
Saito, A,
Pietromonaco S,
Loo AK,
and
Farquhar MG.
Complete cloning and sequencing of rat gp330/"megalin," a distinctive member of the low density lipoprotein receptor gene family.
Proc Natl Acad Sci USA
91:
9725-9729,
1994
35.
Schell, MJ,
Maurice M,
Stieger B,
and
Hubbard AL.
5' Nucleotidase is sorted to the apical domain of hepatocytes via an indirect route.
J Cell Biol
119:
1173-1182,
1992[Abstract].
36.
Simons, K,
and
Wandinger-Ness A.
Polarized sorting in epithelia.
Cell
62:
207-210,
1990[ISI][Medline].
37.
Swiatecka-Urban, A,
Duhaime M,
Coutermarsh B,
Karlson KH,
Collawn J,
Milewski M,
Cutting GR,
Guggino WB,
Langford G,
and
Stanton BA.
PDZ domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem
277:
40099-40105,
2002
38.
Takeda, T,
Yamazaki H,
and
Gist Farquhar M.
Identification of an apical sorting determinant in the cytoplasmic tail of megalin.
Am J Physiol Cell Physiol
284:
C1105-C1113,
2003.
39.
Thomas, DC,
and
Roth MG.
The basolateral targeting signal in the cytoplasmic domain of glycoprotein G from vesicular stomatitis virus resembles a variety of intracellular targeting motifs related by primary sequence but having diverse targeting activities.
J Biol Chem
269:
15732-15739,
1994