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Reciprocal Regulation of and

Costin N. Antonescu1, Timothy E. McGraw2, and Amira Klip3

1Department of Chemistry and Biology, Ryerson University, Toronto, Ontario M5B 2K3, Canada 2Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10065 3Program in Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada Correspondence: [email protected]; [email protected]

The cellular uptake of many nutrients and micronutrients governs both their cellular avail- ability and their systemic . The cellular rate of nutrient or ion uptake (e.g., , Fe3þ,Kþ) or efflux (e.g., Naþ) is governed by a complement of membrane transport- ers and receptors that show dynamic localization at both the plasma membrane and defined intracellular membrane compartments. Regulation of the rate and mechanism of endocytosis controls the amounts of these on the cell surface, which in many cases determines nutrient uptake or secretion. Moreover, the metabolic action of diverse hormones is initiated upon binding to surface receptors that then undergo regulated endocytosis and show distinct signaling patterns once internalized. Here, we examine how the endocytosis of nutrient transporters and carriers as well as signaling receptors governs cellular metabolism and thereby systemic (whole-body) metabolite homeostasis.

nteractions between the cell and its environ- by the balance of its endocytosis and its recy- Iment obligatorily involve events at the plasma cling back to the cell surface from endosomes membrane. Cell-surface proteins mediate nutri- and other intracellular compartments (Fig. 1). ent uptake, product release, and the sensing of Changes in the kinetics of membrane environmental changes, including signals from traffic acutely affect the levels of individual pro- other cells. Appropriate sensing and response to teins at the cell surface and thereby impact how extracellular cues is essential for the individual cells intake nutrients, sense the environment, cell’s survival and for the coordinated cellular and respond to external cues. behavior in multicellular organisms. Accord- Selective molecular mechanisms trigger ingly, maintenance and dynamics of membrane traffic of plasma membrane proteins through proteins are fundamental mechanisms of cellu- endomembranes. Among them, ubiquitination lar homeostasis and survival. and stand out as they can di- Most plasma membrane proteins are in de- rectly target the cargo proteins. Ubiquitination fined equilibria with intracellular endosomal is the covalent attachment of the 76- compartments, such that the amount of a given polypeptide to the 1-amino group protein at the plasma membrane is determined of specific lysine residues (reviewed by Miranda

Editors: Sandra L. Schmid, Alexander Sorkin, and Marino Zerial Additional Perspectives on Endocytosis available at www.cshperspectives.org Copyright # 2014 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a016964 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016964

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Nutrients and hormones Cell surface

Transporters and receptors

Exocytosis/ Endocytosis recycling

Endosomes

Figure 1. Dynamic regulation of the cell-surface content of membrane proteins. Integral membrane proteins found at the cell surface are dynamically localized to the plasma membrane. The amount of any of these proteins at the cell surface is the result of the balance of or recycling of vesicles containing that protein from intracellular membrane compartments and the endocytosis of the protein from the cell surface. Regulation of either the rate of exocytosis or endocytosis results in alteration of the cell-surface content of a given protein.

and Sorkin 2007; and see also Piper et al. 2014). affinity chelating glycoprotein made by the Ubiquitination of cell-surface proteins is the liver (Gkouvatsos et al. 2012). Endocytosis of principal mechanism of control of endocytosis diferricTfistheprincipalmechanismforcellular in yeast (MacGurn et al. 2012), whereas in mam- Fe3þ uptake. receptor 1 (TfR1), a mals, additional molecular mechanisms regu- homodimeric type II 3þ late the endocytosis of cell-surface proteins, in- that binds Tf-Fe2 with nanomolar affinity, is cluding alterations in conformation that impact the primary receptor mediating cellular uptake 3þ interaction with other proteins, and as men- (Fig. 2). The Tf-Fe2 :TfR1 complex is internal- tioned, phosphorylation. Each of these modifi- ized by clathrin-mediated endocytosis (CME) cations can either enhance or reduce the rates (Dautry-Varsat et al. 1983; Klausner et al. internalization, recycling, or degradation of spe- 1983). At the acidic luminal pH of endosomes cific proteins, highlighting the complexity of the (pH 5.5–6.0), Fe3þ is released from Tf, reduced, regulation of endomembrane traffic. The intri- and transported to the cytosol. Apo-Tf (iron- cate mechanisms that underlie the reciprocal free) has high affinity for TfR1 at acidic pH and regulation of endocytosis and metabolism are therefore remains bound to TfR1 in endosomes. beginning to be understood. Here we discuss The Apo-Tf:TfR1 complex returns to the plasma the endocytosis mechanisms in the regulation membrane through mildly acidic, endocytic re- of cellular intake or efflux of iron, cholesterol, cycling compartments. Apo-Tf has an approxi- Naþ, and glucose, and in the regulation of re- mate 500-foldloweraffinityforTfR1atneutral ceptor signaling relevant to metabolism. pH and it is therefore released from TfR1 follow- ing the fusion of recycling vesicles with the plas- ENDOCYTOSIS IS REQUIRED FOR THE ma membrane (where it is exposed to extracel- UPTAKE OF CIRCULATING NUTRIENT lular milieu of pH 7.4). The released apo-Tf is 3þ CARRIERS free to bind Fe in the blood and mediate addi- tional Fe3þ delivery upon binding to TfR1. Regulation of Iron Homeostasis by TfR1 constitutively cycles between the plas- Transferrin Internalization ma membrane and endosomes (Watts 1985; Gi- Ferric iron (Fe3þ) is an essential nutrient that is rone`s and Davis 1989). Internalization of TfR1 carried in the blood by transferrin (Tf ), a high- is mediated by a cytoplasmic YTRF-based motif

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Endocytosis and Metabolism

Fe3+

Cell surface

CME

3+ Fe Recycling endosome

Tfn receptor Early endosome Holo-Tf Apo-Tf

Figure 2. The uptake of iron through endocytosis and recycling of transferrin. Iron-bound transferrin (holo-Tf ) binds to the (TfR) at the cell surface. TfR constitutively internalizes via clathrin-mediated endocytosis. Arrival of the Tf/TfR complex to the acidic environment of endosomes results in dissociation of iron from Tf. This is followed by recycling of the apo-Tf/TfR complex to the cell surface, where apo-Tf dissociates from TfR. The iron is thereby retained intracellularly, and Tf and TfR are able to participate in additional rounds of iron uptake.

associating with the m-subunit of the AP2 rects the receptor to degradation in lysosomes clathrin adaptin complex (Jing et al. 1990; (Tachiyama et al. 2011). Together, these mecha- McGraw and Maxfield 1990; Ohno et al. 1995; nisms control less Fe3þ accumulation upon ele- Owen and Evans 1998). Mutations in YTRF re- vated cellular Fe3þ. ducetheinternalizationrateconstantoftheTfR1 Whole body iron homeostasis is achieved and correspondingly reduce the rate of Fe3þ in- by the balance of TfR1-mediated uptake from tracellularaccumulation, revealingthatinternal- duodenal (dietary iron) and mac- ization of the TfR1 is rate limiting for Fe3þ up- rophages (iron recycled from erythrocytes) to take (McGrawand Maxfield 1990; Pytowski et al. the plasma via the (FPN)-depen- 1995). dent iron transport (Donovan et al. 2005). Although both constitutive endocytosis and The amount of FPN at the surface of these cells recycling of TfR1 are key elements controlling determines the rate of iron export to plasma. cellular Fe3þ accumulation by maintaining the , a liver-produced, 27-amino acid hor- appropriate steady-state level of TfR1 at the plas- mone, controls iron transport from enterocytes ma membrane, the regulation of TfR1 traffic and into the plasma by inhibiting (endocytosis and/or recycling) is not a major FPN. Hepcidin binds FPN inducing its ubiqui- mode for the modulation of cellular of Fe3þ ac- tylation-dependent endocytosis (Nemeth et al. cumulation. Rather, regulation of iron accumu- 2004; Qiao et al. 2012). Internalized FPN is tar- lation is controlled at the level of TfR1 mRNA geted to lysosomes for degradation. Although stability (Rouault 2006). In low Fe3þ conditions the molecular details of the ubiquitin-based there is increased TfR1 mRNA stability and FPN internalization and down-regulation have elevated total TfR1 protein that brings about not been fullyelucidated, it is clear that hepcidin a steady-state increase of TfR1 at the plasma regulation of FPN endocytosis is essential for membrane and augmented capacity for Tf up- iron homeostasis, because mutations that affect take. Conversely, with elevated Fe3þ there is re- hepcidin-FPN binding or FPN ubiquitylation duced TfR1 translation. In addition, increased cause diseases in animal models Fe3þ results in TfR1 ubiquitination, which di- and humans (De Gobbi et al. 2002). Thus, reg-

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ulation of FPN endocytosis is a physiologically distinct lipoproteins. Interestingly, LDL uptake important example of the acute regulation of a but not that of VLDL by LDLR involves ARH nutrient carrier by endocytosis. S-nitrosylation at C199 and C286 (Zhao et al. 2013), required for ARH interaction with AP2. This suggests a mechanism by which LDLR-de- Low-Density Lipoprotein Receptor Endocytosis pendent cellular internalization of LDL can be Facilitates Cellular Cholesterol Uptake separately regulated from that of VLDL, there- Cellular cholesterol uptake occurs through by defining a mechanism by which the systemic binding of low-density lipoprotein (LDL) par- metabolism of these different lipoproteins can ticles by the LDL receptor (LDLR), followed by be distinctly regulated. receptor endocytosis. LDLR binds apo-B100, Strikingly, of the nearly 1000 mutations in which is present in a single copy within each LDLR linked to genetic hypercholesterolemia LDL particle (Jeon and Blacklow 2005; Gold- (Leigh et al. 2008), several impair LDLR inter- stein and Brown 2009). Upon internalization, nalization but not LDL binding (Hobbs et al. the lower endosomal pH causes dissociation 1990). In particular, one of the first monoge- of LDL from its receptor. LDLR recycles back netic mutations identified in LDLR leading to to the cell surface (Brown et al. 1983), whereas hypercholesterolemia was Y807C; thus mutated LDL continues to lysosomes, where cholesterol LDLR shows markedly lower recruitment of is released and rerouted. Cholesterol uptake by LDLR to clathrin-coated pits and virtually ab- this mechanism also suppresses endogenous lated LDLR endocytosis (Davis et al. 1986). cholesterol biosynthesis. Moreover, mutations in ARH that impair LDLR internalization occurs via CME (Jeon LDLR internalization also lead to autosomal- and Blacklow 2005; Goldstein and Brown 2009). recessive hypercholesterolemia (Soutar et al. Early studies identified aromatic-based endo- 2003). Collectively, these studies reveal a critical cytic sorting motifs on LDLR (Davis et al. contribution of LDLR endocytosis to systemic 1987) and the 802FDNPVY807 sequence was later cholesterol homeostasis (Bonfleur et al. 2010). shown to act as a recognition motif for the CME adaptor protein ARH (Traub 2003). Interesting- ly, LDLR endocytosis by CME requires the en- ENDOCYTOSIS LIMITS AND Naþ EFFLUX THROUGH MEMBRANE docytic adaptor proteins ARH or Dab2, which TRANSPORTERS are dispensable for internalization of other CME cargo such as TfR1 (He et al. 2002; Maurer In contrast to the cellular internalization of iron and Cooper 2006). and cholesterol carriers described above, other LDL particles are 22 nm in diameter, mak- nutrients and micronutrients are taken up di- ing each LDL-LDLR complex significantly larg- rectly through membrane-bound transporters er than most CME cargo. Expression of a chi- straight into the cytosol. Efflux also occurs di- meric protein harboring the cytoplasmic region rectly from the cytosol to the extracellular mi- of LDLR results in ARH- and Dab2-dependent lieu across membrane transporters. The rate of changes in the size of clathrin-coated pits, even nutrient or micronutrient uptake/efflux is in the absence of LDL (Mettlen et al. 2010). This largely determined by the permanence of their indicates that incorporation of LDLR-ARH/ cognate transporters at the cell surface, which is Dab2 complexes into endocytic structures may fundamentally controlled by the balance of their “customize” CME to accommodate these larger endocytosis and recycling. cargoes. LDLR also contributes to the internalization GLUT Endocytosis Limits the Rate of Cellular of other lipoproteins, including very-low-den- Glucose Uptake sity lipoproteins (VLDL). This raises the ques- tion of how the internalization of a single recep- A family of facilitative glucose transporters, tor may regulate the unique homeostasis of GLUTs, is responsible for the transport of glu-

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Endocytosis and Metabolism

cose from the blood into cells. The 14 different types. Glucose uptake through GLUT1 is regu- GLUTs vary in their tissue expression and intrin- lated by changes in the individual transporter sictransport parameters such asturnover number efficiency (“transport activity,” such as by intra- (Mueckler and Thorens 2013). Here we discuss cellular ATP levels) (Blodgett et al. 2007) and in the contribution of endocytosis of two of them, the number of transporters at the plasma mem- GLUT1 and GLUT4, to the regulation of glucose brane. Here we focus on the latter mechanism as transport. Notably, each of these transporters a means to regulate glucose flux. cycles differentially within the cell, and indeed In hematopoietic cells, cytokines (e.g., in- their rates of endocytosis differ substantially: terleukin 3) promote glucose uptake by raising Whereas GLUT4 is rapidly removed from the the amount of GLUT1 at the plasma membrane cell surface to be stored in still poorly described (Kan et al. 1994; Rathmell et al. 2001). This but highly dynamic storage vesicles, GLUT1 has increase is achieved via cytokine-induced inhi- a longer permanence at the cell surface. Recent bition of GLUT1 internalization from the mem- studies have discovered that the endocytosis of brane and stimulation of GLUT1 translocation each of these transporters is exquisitely regulat- from intracellular compartments to the mem- ed. Brief examples are presented next. brane (Fig. 3) (Wieman et al. 2007; Wofford et al. 2008). Upon cytokine withdrawal, GLUT1 internalization is accelerated and recycling to GLUT1 the plasma membrane inhibited, resulting in GLUT1, the most widely expressed GLUT iso- an acute, net reduction of GLUT1 at the plasma form, mediates glucose uptake into most cell membrane and a corresponding drop in glucose

Glucose

GLUT1 GLUT4 Insulin CIE AMPK and energy AMPK, TXNIP CME stress contraction Cytokines (via Akt)

GLUT4 storage Endosomes compartment GLUT1 GLUT4 Tfn rec. VAMP2 IRAP

Figure 3. Control of glucose uptake by regulation of GLUT1 and GLUT4 endocytosis. Glucose transport from the extracellular milieu to the occurs selectively by glucose transporters (GLUTs) present at the cell surface. GLUT4 endocytosis occurs through both clathrin-mediated (CME) as well as clathrin-independent (CIE) endocytosis. The cell-surface content, and hence the rate of glucose uptake by GLUT1 and GLUT4, is regulated by alterations in the endocytosis of these glucose transporters, as well as the rates of recycling. Moreover, GLUT1 and GLUT4 undergo distinct intracellular sorting, with 50% of GLUT4 residing in a specialized storage compartment. Shown are some of the stimuli and intracellular signals that reduce the endocytosis of GLUT1 and GLUT4 or increase transporter recycling and thereby increase the rate of cellular glucose uptake. Also shown are the insulin-responsive aminopeptidase (IRAP) and VAMP2,which are enriched in the GLUT4 storage compartment and the transferrin receptor (Tfn rec.), which is not, and is instead found largely in recycling endosomes.

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C.N. Antonescu et al.

transport. The molecular mechanism for cyto- generation ensue. Further, TXNIP also reduces kine-induced changes in GLUT1 traffic has not GLUT1 transcription, contributing to the long- been fully described. It is known that it occurs term reduction in glucose uptake. downstream from the Akt kinase, because en- forced expression of constitutively active Akt GLUT4 prevents enhanced GLUT1 internalization and reduced recycling upon growth factor with- GLUT4 expression is mostly limited to muscle drawal (Wieman et al. 2007). and adipose cells (Mueckler and Thorens 2013) In a different physiological context, cellular and is responsible for most of the glucose re- energy deprivation enacts an emergency re- trieval from blood following a meal. In the fast- sponse that increases glucose uptake. This is in ed state (between meals) glucose transport into part mediated by an increase in surface GLUT1, adipose and muscle cells is low because 90%– which is in the short run mediated by inhibition 95% of GLUT4 is sequestered intracellularly of its constitutive endocytosis, and in the long (Li et al. 2001; Huang and Czech 2007). Insulin, run by enhanced GLUT1 synthesis. The ele- which increases in the blood upon feeding, ments participating in the acute response were stimulates a rapid (within minutes) redistribu- recently found to involve the arrestin-domain- tion of GLUT4 from intracellular compart- containing protein (ARRDC), thioredoxin-in- ments to the plasma membrane, causing a con- teracting protein (TXNIP). TXNIP expression comitant surge in glucose transport into these correlates with glucose transport and its knock- tissues (Foley et al. 2011; Rowland et al. 2011). down in adipose and muscle cells lowered glu- The elevated rate of glucose transport thus cose uptake (Parikh et al. 2007). TXNIP affects achieved is behind the return of blood glucose GLUT1 traffic (Wu et al. 2013), by forming a levels back to prefeeding levels within 1–2 h complex that promotes its internalized through (Ferrannini et al. 1985). The redistribution of clathrin-coated pits. Although ARRDCs are GLUT4 to the plasma membrane is reversible. involved in the ubiquitination of membrane When blood insulin levels wane, GLUT4 redis- proteins (MacGurn et al. 2012), ubiquitination tributes back from the plasma membrane to in- of GLUT1 has not been reported, and it is tracellular compartments. Unlike intracellular not known if TXNIP controls GLUT1 endocy- GLUT1, which transits only through recycling tosis by ubiquitination of another protein. The endosomes, GLUT4 is unique in its sorting out TXNIP-induced drop in surface GLUT1 reduces of recycling endosomes into the TGN and a spe- glucose transport. cific “GLUT4 storage” compartment. The na- TXNIP transcription is under the regulation ture of this compartment is highly debated, of a number of different glucose-sensing tran- but it is apparent that it excludes certain pro- scription complexes, CHREBP-Mlx and Mon- teins such as GLUT1 and most of the TfR1, doA-Mlx, such that TXNIP mRNA levels in- whereas it is enriched in proteins such as crease with increasing glucose uptake. Because VAMP2and IRAP (Fig. 3) (Jedrychowski et al. TXNIP has an inhibitory effect on glucose up- 2010; Foley et al. 2011; Sto¨ckli et al. 2011; Leto take, the glucose-stimulated increased TXNIP and Saltiel 2012). Most studies agree that the mRNA provides a negative-feedback loop for GLUT4 storage compartment is distinct from GLUT1-mediated glucose uptake. In addition, the TGN although it contains proteins such as TXNIP phosphorylation by AMP-dependent Syntaxin-6 and -16, and these may contribute to protein kinase (AMPK) promotes its degrada- its sorting or functionality. Sorting to this selec- tion (Wu et al. 2013). AMPK is activated when tive compartment, unique to fat and muscle the AMP/ATPratio increases, therefore AMPK- cells, may prevent GLUT4 from degradation induced degradation of TXNIP will result in and may account for its strikingly long half- elevated plasma membrane GLUT1 as a conse- life (nearly 48 h). The localization of this unique quence of reduced internalization, and a corre- compartment is also poorly defined, in part ow- sponding increase of glucose uptake and ATP ing to spatial constraints in adipose and mature

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Endocytosis and Metabolism

muscle cells, and to its dynamic nature. Howev- way that is independent of AP2 or any of the er, there is general agreement that GLUT4 se- known GLUT4 traffic mo- questration is essential for its appropriate re- tifs (Blot and McGraw 2006). In unstimulated sponse to insulin and hence defining the full L6 muscle cells, GLUT4 internalizes via both complement of signals dictating the dynamic a clathrin-dependent and a cholesterol and retention of GLUT4 in intracellular stores is of dynamin-dependent but caveolin-independent utmost physiological importance. mechanism (Antonescu et al. 2008a, 2009). Ki- netically, GLUT4 endocytosis differs from that of the TfR1 or the receptor for a2-macroglobu- GLUT4 Internalization in the Basal State lin (Habtemichael et al. 2011), markers of CME GLUT4 is a very long-lived protein; hence, each and non-CME, noncaveolar internalization. molecule can undergo numerous cycles of in- ternalization and recycling. The pronounced GLUT4 Internalization in Response intracellular GLUT4 sequestration in unstimu- to Insulin lated fat and muscle cells is owing to its rapid internalization and slow recycling. GLUT4 con- In muscle and fat cells the predominant effect of tains sequences that determine its endocyto- insulin is enhanced recycling (i.e., re-exocyto- sis and intracellular sorting. When ectopically sis) of GLUT4 to the plasma membrane (Li et al. expressed in fibroblast-like cells, both the phe- 2001; Karylowski et al. 2004; Martin et al. 2006; nylalanine-based motif FQQI on the GLUT4 Blodgett et al. 2007; Antonescu et al. 2008b, amino-terminal cytoplasmic domain and a di- 2008a; Ishikura et al. 2010), and this is also the motif on its carboxy-terminal cytoplas- case in (Karlsson et al. 2009). In mic domain function as internalization motifs , insulin also slows down GLUT4 in- (Corvera et al. 1994; Garippa et al. 1994, 1996; ternalization (Fig. 3) (Czech and Buxton 1993; Verhey et al. 1995). These motifs suffice to pro- Blot and McGraw 2006), but this does not occur mote internalization when transferred to other in either cardiomyocytes (Yang and Holman proteins, and belong to the two major families 2005) or L6 muscle cells (Li et al. 2001; An- of membrane protein sorting signals: aromatic- tonescu et al. 2008a, 2009) (although a report based motifs and dileucine-based motifs (Sea- to the contrary exists [Fazakerley et al. 2010]). man 2008; see also Traub and Bonifacino 2013). Once internalized, insulin enhances the rate of However, the aromatic-based motif on GLUT4 GLUT4 transit through recycling endosomes has a instead of the more com- (Foster et al. 2001). Although the magnitude mon tyrosine. Substitution of the endogenous of the effect of insulin on recycling is greater phenylalanine for tyrosine in the GLUT4 mo- than on internalization, both changes contrib- tif promotes faster (2) internalization of the ute significantly to the net redistribution of transporter (Garippa et al. 1996; Blot and GLUT4 to the plasma membrane. This may be McGraw 2006). Similarly phenylalanine-based one reason why the insulin-dependent net gain motifs on other proteins promote a slower in- in surface GLUT4—and its consequent glucose ternalization compared with tyrosine-based uptake—is larger in adipocytes than in muscle motifs (e.g., McGraw and Maxfield 1990). The or heart cells. The molecular basis of insulin- behavior of GLUT4 in fibroblast-like cells is induced reduction in GLUT4 internalization consistent with its internalization by a cla- in adipocytes is beginning to be understood. thrin/AP2-dependent pathway, although the Under this stimulus, the FQQI motif functions mechanism of internalization of ectopically ex- as the main GLUT4 internalization motif, pro- pressed GLUT4 endocytosis in these cells has not moting uptake via an AP2-dependent mecha- been rigorously investigated. In contrast, more nism (Blot and McGraw 2006). Thus, the data recent studies in 3T3-L1 adipocytes reveal that, suggest that the GLUT4 internalization route in the basal state, GLUT4 is internalized by a varies between insulin-stimulated and -unstim- cholesterol-dependent, nystatin-sensitive path- ulated adipocytes (respectively, AP2-depen-

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C.N. Antonescu et al.

dent and -independent). How this change in (Antonescu et al. 2008a) slows the rate of route is achieved is unknown. By comparison, GLUT4 endocytosis (Fig. 3). DNP causes an GLUT4 internalizes through both clathrin-de- initial and abrupt drop in cellular ATP with pendent and clathrin-independent routes in concomitant activation of AMPK; within min- both basal and insulin-stimulated muscle cells, utes, intracellular ATP levels bounce back, coin- and insulin does not appear to change the pro- cident with the AMPK-dependent increase in portion or rate of internalization of either route cell surface GLUT4 elicited by DNP (Klip et al. (Antonescu et al. 2008a, 2009). GLUT4 inter- 2009). Thus, AMPK reduces GLUT4 endocyto- nalization appears to require the F-BAR-do- sis to enhance GLUT4 permanence at the mem- main-containing protein CIP4, perhaps to brane. Indeed, activation of AMPK by treatment cause membrane curvature for formation of en- of L6 muscle cells with the AMPK-activator docytic vesicles (Hartig et al. 2009; Feng et al. AICAR alone (Fazakerley et al. 2010) or AICAR 2010). in combination with exogenous activation of What is the significance of the different protein kinase C (Antonescu et al. 2008a) low- internalization routes? Although this is still an ered the rate of GLUT4 endocytosis. Other ex- open question, one can make certain testable amples of reduced GLUT4 endocytosis upon predictions: If the cholesterol-dependent route exposure of energy stressors include the re- is faster than the FQQI-dependent one, the in- sponse of adipocytes to dimethyl sulfoxide (Be- sulin-dependent shift to clathrin/AP2-mediat- renguer et al. 2011), rosiglitazone (Velebit et al. ed internalization through the FQQI motif in 2011), (Yang and Holman 2006), adipocytes might allow for longer permanence and chronic insulin (Pryor et al. 2000). at the plasma membrane of each transporter— In physiological conditions, muscle contrac- to enact more rounds of glucose transport— tion vigorously uses ATP that must be replen- before being internalized. This would be facili- ished by glucose influx. Contraction increases tated by the slower internalization conferred surface GLUT4 levels in skeletal muscle (Douen to GLUT4 by the FAAI motif (compared with et al. 1990; Goodyear et al. 1991) and this occurs the canonical YQQI motif ). The slower inter- through both AMPK- and Ca2þ-dependent sig- nalization would be compounded by the gain in nals. Interestingly, in noncontracting muscle transporters at the membrane resulting from in- cells, a depolarization-induced influx of Ca2þ creased recycling. In muscle, the clathrin-de- suffices to reduce GLUT4 internalization, and pendent pathway operating in the basal state this response is independent of AMPK avail- might confer a slower internalization that could ability (Wijesekara et al. 2006). Moreover, a cal- be advantageous for its physiological needs. cium ionophore, ionomycin, can slow down the rate of GLUT4 endocytosis and promote GLUT4 exocytosis, but only the latter effect GLUT4 Internalization in Response is AMPK-dependent (Q Li, P Bilan, W Niu, to Energy Demand et al., unpubl.). The exocytic increase in surface Processes that increase energy demand require GLUT4 in exercised muscle is also AMPK de- an adaptive increase in carbon source to sustain pendent (Lauritzen et al. 2010). Intriguingly, cellular viability (Cartee et al. 1991). This adap- the AICAR had only a minor effect on GLUT4 tive response in other cells is ascribed to GLUT1 exocytosis in skeletal muscle (Karlsson et al. (see above), but muscle largely depends on 2009), suggesting that AMPK may instead act GLUT4 for influx of glucose. In contrast to in- on the endocytic step and/or that it may act in sulin stimulation, energy status signaling leads concert with other physiological signals to in- to a marked slowdown of GLUT4 endocytosis in crease surface GLUT4 during muscle contrac- muscle cells. Metabolic stress resulting from tion. Hence, AMPK regulates GLUT4 endocy- treatment of cardiomyocytes with metformin tosis in conditions of energy demand (e.g., DNP or oligomycin (Yang and Holman 2005) or of treatment) and GLUT4 exocytosis in response L6 muscle cells with 2,4-dinitrophenol (DNP) to Ca2þ-dependent signals. Collectively, these

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studies show that signals elicited by alterations glycosylated b subunit. The 110-kDa a sub- in energy status and during skeletal muscle con- unit has 10 transmembrane segments, whereas traction converge to regulate the rates of GLUT4 the 31.5 kDa b subunit crosses the membrane endocytosis and exocytosis, thereby enhancing once. The ab heterodimer is the stable form, as intake of energy-providing glucose. changes in copy number of the a subunit re- quire concomitant changes in the b subunit to create functional units. Depending on the Na/K-ATPase Endocytosis Controls cell type, about 800,000 to 30 million pump Multiple Metabolic Functions copies are present at the cell surface at any given In all mammalian cells, an electrochemical gra- time. Abnormal changes in the copy number of dient—creating a voltage difference that is typ- pump units occur in several pathologies such ically negative with regards to the external mi- as heart failure and hypertension associated lieu—ensures proper movement of ions and with excessive renal Naþ resorption. The func- regulation of cell volume. At the core of the tional pump involves one of four isoforms of the maintenance of this electrochemical Naþ gradi- catalytic a subunit and one of any of the three ent lays the Naþ/Kþ-ATPase or Naþ/Kþ pump. isoforms of the stabilizing b subunit. The By expending one ATP molecule per cycle of transcription and translation control of each of efflux of three intracellular Naþ ions and influx these subunits is tissue specific and differentially of two extracellular Kþ ions, the pump ensures regulated by diverse physiological conditions, as rapid riddance of any Naþ ions that increase in is the catalytic activity of the a subunit. On top the cytosol as a result of numerous Naþ-depen- of that, the regulation of pump availability at dent influx processes. Such preceding influx the cell membrane through exocytosis and en- processes are key for lowering intracellular docytosis is afundamental physiological control Ca2þ (Naþ/Ca2þ exchange), Naþ-coupled ami- mechanism. no acid transport, Naþ-coupled glucose uptake in epithelial cells, and for a diversity of Naþ,Kþ Pump Regulation by Exocytosis and Cl2 transport processes. In fact, the Naþ/ Kþ pump is the main, if not only, mechanism of The increase in pump surface exposure is best Naþ efflux from most cells. In parallel, the exemplified by the increase in surface a2 and b1 pump returns Kþ to cells that release or leak subunits that occurs in skeletal muscle in re- this ion in the course of their electrical activity, sponse to insulin (Hundal et al. 1992; Ewart such as during action potentials in neurons, and Klip 1995; Benziane and Chibalin 2008). skeletal, smooth and heart muscles, and neuro- This rapid up-regulation of surface pump num- endocrine cells. Another fundamental function beroccurs independently of biosynthetic chang- of the pump is the translocation of Naþ from es and although typically ascribed to translo- one side of epithelia to the other, that along with cation to the membrane, the endocytic and obligatory anion movement generates the os- exocytic arms of pump traffic in response to motic gradient that drives water absorption or insulin have not been directly measured. A rapid resorption. increase in surface pump units is vital to manage With this tall order, it is not surprising that the rapid uptake of dietary Kþ (along with the the activity of the Naþ/Kþ pump is rapidly and Naþ,Kþ,Cl2 ) and importantly exquisitely regulated. This is achieved by chang- handles the elimination of cytosolic Naþ excess es in both the activity and the availability of the that results from Naþ-coupled amino acid up- pump at the surface of the cell in question. take and Naþ/Hþ exchange promoted by the hormone. A similar up-regulation occurs in the contracting skeletal muscle fiber. In the cen- Structural Properties of the Naþ/Kþ Pump tral nervous system, the pump is up-regulated The pump consists of a heterodimer of one on demand to mediate Kþ reuptake and rids catalytic a subunit and one stabilizing, highly neurons of Naþ gained during electrical activity.

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Naþ/Kþ-Pump Endocytosis Triggered phorylation of Ser-18 on the a1 subunit of the þ þ by CO2 and Hypoxia in the Lung Na /K pump (Gusarova et al. 2009). This is thought to lead to ubiquitylation of a lysine Changes in CO2 and O2 tension occur contin- residue in the vicinity of Ser-18 as preamble to uously in the lung, and when these changes are the pump endocytosis (Dada et al. 2007). This extreme, fluid movement needs to be regulated. mechanism is partly reenacted in the kidney in This is enacted primarily through changes in response to elevated Naþ in the basolateral mi- the availability of the Naþ/Kþ pump at the al- lieu (see below). veolar cell surface, as the pump is the driving Finally, mitochondrial ROS are generated in force for ion-accompanying fluid movement the course of hypoxia and these have been impli- (Bertorello and Sznajder 2005). Elevations in cated in the endocytic regulation of the pump CO2 (hypercapnia) and reductions in O2 (hy- (Vada´sz et al. 2005), apparently through phos- poxia) demand an adaptive reduction in Naþ phorylation of the AP2 adaptin m2 subunit. flux through the cells, and this is achieved by The responsible kinase remains to be identified, reducing the number of Naþ/Kþ pumps at the although AAK1 (adaptor-associated protein cell surface, through their rapid endocytosis. In kinase 1) has been suggested (Chen et al. 2006). fact, the endocytosis of the pump in alveolar epithelial cells controls whole body CO2 clear- ance and alveolar fluid resorption. The reduc- Naþ/Kþ-Pump Endocytosis Triggered tion in pump units at the cell surface also curbs by Naþ, Dopamine, and Cardiotonic ATP utilization, given the high energetic de- Steroids in the Kidney mand imposed by pump activity (up to 30% In the proximal tubule of the kidney, the Naþ/ of the alveolar cell ATP). This reduction is ben- Kþ pump is the driving force for Naþ and fluid eficial in conditions of hypoxia. However, en- recovery from lumen to blood (Naþ resorp- docytosis of the pump can result in decreased tion). The kidney expresses the a1- and b1-sub- alveolar fluid clearance. unit isoforms that are abundant at the baso- The lung preferentially expresses the a1 and lateral membrane, from where the dimer is b1 subunits. The endocytosis of pump subunits endocytosed in response to several stimuli (Ber- at the basolateral surface of primary and im- torello and Sznajder 2005). mortalized alveolar cells (e.g., human A549, The presence of elevated Naþ in the blood rat ATII0) has been recorded using cell-surface and therefore at the peritubular interstitium of biotinylation of proteins followed by pulldown the proximal tubule demands a reduction in with streptavidin beads. Pump endocytosis Naþ resorption from the lumen. High levels of is promoted by CO and by hypoxia, but also 2 Naþ cause release of dopamine, a natriuretic by thrombin (Vada´sz et al. 2005). The signals hormone that increases Naþ excretion by dimin- regulated this endocytic movement are only ishing its reabsorption, primarily in the proxi- beginning to be identified, but hypercapnia mal tubule. Dopamine is synthesized within the activates ERK (extracellular signal-regulating kidney from L-dopa, and both local and circu- kinase) and inhibiting ERK reduces pump en- lating dopamine binds and activates DA1 and docytosis (Welch et al. 2010). On the other DA2 receptors that generate cyclic AMP. Dopa- hand, hypoxia increases CaMKK-b (/ mine triggers Naþ/Kþ-pump endocytosis at calmodulin-dependent protein kinase kinase the basolateral membrane of kidney epithelial 2) and ROS (reactive oxygen species), which cells through three coincident events, docu- activate AMPK (Vada´sz et al. 2008). Indeed, in- mented in OK (opossum kidney) cells (Chibalin hibiting either one of these inputs reduces hy- et al. 1999) and renal proximal tubule cells (Pe- poxia-induced pump endocytosis. It has been demonte et al. 2005): proposed that AMPK phosphorylates the atyp- ical protein kinase C-z on Thr-410, activating 1. Phosphorylation of Ser-18 on the a1 subunit it, and that this signaling pathway leads to phos- of the Naþ/Kþ pump (Pedemonte et al.

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Endocytosis and Metabolism

2005), mediated by protein kinase C-z. This through sparing phosphorylation of the Rab- promotes association of dynamin-2 with the GAP AS160 (thereby maintaining it in its a subunit (Efendiev et al. 2002). Presumably, GAP-active form) (Alves et al. 2010). This sug- phosphorylation of dynamin-2 at position gests that AS160, known to retain GLUT4 in S848 limits basal Naþ/Kþ-pump endocy- intracellular stores, also retains the pump intra- tosis and dopamine-regulated PP2 dephos- cellularly, effectively preventing it from recy- phorylation of dynamin-2 S848 promotes cling to the cell surface. Naþ/Kþ-pump internalization (Efendiev et Finally, additional to the Naþ-dependent al. 2002). release of dopamine that regulates the pump through GPCRs, basolateral Naþ appears to 2. Simultaneously, the a1 subunit is also phos- cause release of cardiotonic steroids (endoge- phorylated on Y537, promoting binding of nous, digitalis-like factors), which act on the the p85 subunit of phosphatidylinositol-3- proximal tubule cells, sending signals that pro- kinase (PI3K). Both phosphorylation events mote Naþ/Kþ-pump endocytosis (Bagrov et al. of a1 on Ser-18 and Y537 are required for its 2009). In cell cultures, these endogenous car- endocytosis (Done´ et al. 2002). Binding of diotonic steroids are emulated by using oua- PI3K to the pump occurs via interaction of bain (irrespective of its inhibitory action on the SH3 domain of p85 with a proline-rich pump activity). The ouabain-induced Naþ/ domain of the a1 subunit, a region of the Kþ-pump endocytosis requires clathrin (Liu pump distinct from that bound by AP2 (Yu- et al. 2004) although the same group also re- dowski et al. 2000). In fact, PI3K activity ported endocytosis through a cholesterol- and enhances association of AP2 with the a1 caveolin-1-dependent route (Liu et al. 2005). subunit (Yudowski et al. 2000). This results Clearly, more work is required to elucidate the in formation of a complex of the Naþ/Kþ endocytic route involved in the response to oua- pump, AP2, clathrin, and dynamin-2 (Pede- bain and, if more than one, it is paramount to monte et al. 2005). identify which one is regulated, and how. Addi- 3. Finally, the adaptin m2 subunit is phosphor- tional regulatory factors act on the pump, such ylated on T156 in response to dopamine and as prostaglandins, reduce Naþ/Kþ-pump ac- ROS (Chen et al. 2006), and m2 binds di- tivity in the collecting renal tubular cells, and rectly to the YLEL motif on the main cyto- it will be important to find if this effect is also plasmic loop of the Naþ/Kþ pump (Done´ mediated by pump endocytosis. et al. 2002; Chen et al. 2006). The pump then Collectively, the above examples illustrate apparently internalizes all the way to late the variety of stimuli that induce Naþ/Kþ- endosomes (based on subcellular fraction- pump endocytosis and the diverse intracellu- ation experiments) from whence it can recy- lar mechanism that participate in this phenom- cle to the membrane (Chibalin et al. 1999). enon.

In addition to this interesting model, other elements contribute to pump endocytosis in RECIPROCAL REGULATION OF METABOLISM, ENDOCYTOSIS, epithelial cells following activation of G-pro- AND RECEPTOR SIGNALING tein-coupled receptors (GPCRs) by dopamine, vasopressin, or adrenergic hormones. In COS Hormones and growth factors that are recog- cells (a monkey kidney cell line), Naþ/Kþ- nized by surface receptors have evolved mecha- pump endocytosis involves its direct phosphor- nisms to signal differentially from the plasma ylation through GPCR-associated kinases 2 membrane and endocytic locales (see Di Fiore and 3 and binding of arrestin 2 and 3 (Kimura and von Zastrow 2014). This applies particular- et al. 2007). In MDCK (Madin-Darby canine ly to receptor tyrosine kinases (RTKs) and kidney) epithelial cells, inhibition of AMPK GPCRs that impact on protein, lipid, and car- contributes to Naþ/Kþ-pump endocytosis, bohydrate metabolism. Endocytosis is also a

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mechanism to end defined signal transmission. bolic signaling, and (3) insulin degradation to Internalized receptors can then either recycle terminate its action. to the membrane or continue toward degrada- tion. Receptor removal from the membrane, IR Endocytosis in Transendothelial whether for recycling or degradation, can lead Insulin Transport to ligand desensitization, whereas recycling af- fords resensitization. The internalization and Transendothelial insulin transport may repre- postendocytic fate of the receptor-ligand com- sent the rate-limiting step of insulin action in plex governs the magnitude, duration, and spe- vivo (Miles et al. 1995; Barrett et al. 2009), and cificity of receptor signaling (Sorkin and von in aorta endothelial cells occurs through a sat- Zastrow 2009; Kholodenko et al. 2010), and is urable mechanism involving caveolar internali- tailored to conform to the physiological func- zation of insulin-IR complexes (Barrett et al. tion of each hormone. Here we illustrate the 2011). IR internalization and effective insulin endocytic regulation of metabolic signaling by transcytosis in endothelial cells determines in- the insulin, epidermal growth factor (EGF), b2- sulin availability to muscle and fat cells. Surpris- adrenergic, and D2 dopamine receptors. ingly, the molecular underpinnings of this transendothelial transfer remain obscure, pos- sibly due to suitable microvascular endothelial Regulation of Insulin Action by Insulin cell systems. Receptor Endocytosis Insulin, secreted by pancreatic b cells in re- IR Endocytosis Controls Mitogenic but Not sponse to ingested glucose (Rorsman and Ren- Metabolic Signaling stro¨m 2003), binds to insulin receptors (IR) present on its main metabolic target tissues (liv- IR endocytosis is slow in skeletal muscle and fat er, fat, muscle) as well as on epithelial, endothe- cells, but fast in the liver. Upon internalization lial, and neuronal cells. Insulin binding to IR and dissociation from insulin in endosomes, the activates mitogenic signaling (e.g., Shc to Erk), IR retains tyrosine kinase activity (Rosen et al. gene expression (e.g., Foxo), and is a critical 1983; Klein et al. 1987) and activated insulin regulator of systemic carbohydrate and lipid signaling intermediates are associated with en- metabolism (through Akt) (Saltiel and Kahn dosomes in Fao hepatoma and rat liver cells 2001). Insulin-dependent clearance of dietary (Backer et al. 1989; Balbis et al. 2000), adipo- glucose from blood involves the rapid mobiliza- cytes (Kublaoui et al. 1995), and skeletal muscle tion of GLUT4 vesicles described above, which (Dombrowski et al. 2000). Interestingly, block- is regulated by the phosphatidylinositol-3-ki- ing IR internalization impairs insulin-stimulat- nase to Akt axis that activates small G proteins ed Shc and Erk (but not Akt) phosphorylation directing vesicle traffic (Zaid et al. 2008; Foley in hepatoma and CHO cells (Ceresa et al. 1998; et al. 2011). Hamer et al. 2002) but not in 3T3-L1 adipocytes Upon binding insulin, the IR rapidly inter- (Kao et al. 1998). These findings suggest that IR nalizes via CME in rat liver cells (Pilch et al. endocytosis is required for activation of MAPK 1983), 3T3-L1 adipocytes (Fan et al. 1982), in a cell-selective manner, but is not required for mouse embryonic fibroblasts (Morcavallo et al. Akt activation. In fact, inhibiting IRendocytosis 2012), and CHO cells (Paccaud et al. 1992; Car- does not prevent the Akt-dependent GLUT4 pentier et al. 1993), as well as through non- translocation, stimulation of glucose transport, clathrin mechanism(s) in rat adipocytes (Gus- or synthesis in adipocytes (Ceresa et al. tavsson et al. 1999; Fagerholm et al. 2009), 1998; Kao et al. 1998). Consistent with these endothelial cells (Wang et al. 2011), and mouse differential requirements for IR endocytosis of embryonic fibroblasts (Morcavallo et al. 2012). the mitogenic versus metabolic facets insulin IR endocytosis is required for (1) transendo- signaling, the Erk-dependent transcription of thelial insulin transfer, (2) mitogenic and meta- c-fos but not the Akt-dependent regulation of

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the gene by insulin was blocked by Insulin EGF inhibition of IRendocytosis in INS1 cells (Uhles et al. 2007). Hence, the Akt-dependent meta- IR EGFR bolic facets of IR signaling do not require recep- CIE CME tor endocytosis; however, IR endocytosis may provide negative-feedback regulation (Bra¨nn- Recycling mark et al. 2010). Endothelial IDE transcytosis IR Internalization Leading to Insulin Degradation

Up to 50% of insulin secreted into the portal Lysosomal degradation circulation is first degraded by the liver, a pro- cess that requires insulin binding to IR, receptor Figure 4. The internalization and recycling of the internalization, and insulin degradation in en- . Insulin binding to its receptor (IR) dosomes (Valera Mora et al. 2003). Once insulin results in rapid internalization of the insulin-IR com- reaches the peripheral circulation, its levels are plex via either clathrin-mediated endocytosis (CME) or clathrin-independent endocytosis (CIE). On ar- again controlled by uptake and degradation in rival within the acidic environment of endosomes, liver, in addition to regulation of insulin levels insulin dissociates from the IR. Insulin is degraded by uptake in the kidney, muscle, and fat (Valera by insulin-degrading (IDE), whereas the IR Mora et al. 2003). IR endocytosis in the liver undergoes efficient recycling to the PM. In contrast to requires the cardioembryonic antigen-related other tissues, in endothelial cells insulin predomi- cell adhesion molecule 1 (CEACAM1) (Poy et nantly undergoes transcytosis without degradation. al. 2002), whereas in the kidney, insulin binds By comparison, the binding of epidermal growth fac- tor (EGF) to its receptor (EGFR) results in rapid to megalin, and insulin-megalin complexes in- internalization leading largely to lysosomal degrada- ternalize via clathrin endocytosis that proceeds tion of the EGF-EGFR complex. to lysosomal insulin degradation (Orlando et al. 1998). In all instances, including muscle and adipose cells, insulin dissociates from the IR 2009). Down-regulation of EGFR provides a in endosomes (Carpentier et al. 1986) and is limit to the duration of mitogenic signaling. rapidly degraded in by endosomal insulin-de- Although the IR can undergo ubiquitina- grading enzymes (Fig. 4) (Doherty et al. 1990; tion under some circumstances, this does not Tundoet al. 2013). The IR is not degraded and is result in significant IR degradation (Kishi et al. instead rapidly recycled to the membrane (Car- 2007; Song et al. 2013). Insulin dissociation in pentier et al. 1986; Knutson 1991). endosomes is critical for IR recycling, as muta- tions of K460 in IR that impair insulin-IR endo- somal dissociation lead to IR degradation and Inefficient Ligand-Stimulated in vivo (Kadowaki et al. 1990). IR Degradation That the IR is rapidly recycled and not efficient- Compared with the IR, the EGF receptor ly degraded ensures that insulin action is con- (EGFR) is more efficiently degraded upon li- trolled by systemic insulin availability and not gand binding and internalization. EGF stimula- peripheral tissue IR down-regulation. tion activates several prosurvival and mitogenic Hence, EGFR and IR differ in the extent to signaling pathways (Huang et al. 2007). EGF which RTK ubiquitination controls the endo- remains bound to EGFR during endosomal membrane itinerary of the receptor. In addition transit, and this is required to cause EGFR ubiq- to receptor ubiquitination by receptor-proxi- uitination leading to its lysosomal degradation mal signaling events, RTKs may also use recep- (Acconcia et al. 2009), although some EGFR tor-distal signaling to broadly control the mem- recycling also occurs (Fig. 4) (Sorkin and Goh brane traffic of numerous other proteins. A

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recent study in yeast found that activation of 2000; Parent et al. 2009) with minimal receptor mTOR resulted in control of ubiquitin ligase tar- degradation (Tsao and von Zastrow 2000), al- geting by arrestin-related proteins (ARTs, ho- though prolonged adrenergic stimulation caus- mologous to mammalian ARRDCs), which in es b2-AR degradation (Moore et al. 1999). Im- turn led to ubiquitination and internalization portantly, impairment of b2-AR endocytosis of a diverse group of cell-surface proteins (Mac- by perturbation of Arf6 or dynamin selectively Gurn et al. 2011). This work raisesthe possibility blunts adrenergic stimulation of lipolysis (Liu that activation of mTOR by RTKs in mammals et al. 2010). Consistent with a physiological con- mayleadtorobustcontroloftheendomembrane tribution of b2-AR endocytosis to systemic me- traffic and function of a large number of cell- tabolism, polymorphisms of b2-AR (R16G and surface transporters and receptors. Q27E) with defective adrenergic desensitization (Dishy et al. 2001) are linked to differences in fat metabolism and obesity (Ukkola et al. 2000; Regulation of GPCR Signaling by Endocytosis Corbalan et al. 2002). Hence, b2-AR endocy- GPCRs regulate multiple aspects of cellular and tosis is a critical controller of lipolysis, and sug- systemic metabolism. GPCR agonists stimulate gests that either multiple cycles of b2-AR inter- exchange of GDP for GTP on specific Ga sub- nalization and recycling or endosomal-specific units of heterotrimeric G proteins, activating b2-AR signaling are required for stimulation of signaling intermediates including G-protein- lipolysis. coupled receptor kinases (GRKs). GRKs enable binding of b-arrestins to GPCRs, which in turn D2DR Endocytosis Controls Feeding down-regulate G-protein signaling and pro- and Energy Expenditure mote CME of the receptors (Shimokawa et al. 2010). Although GPCR activation of the mito- D2DR is highly expressed in the striatum and genic MAPK pathway begins at the cell surface, nucleus accumbens regions of the brain, which signaling continues within endosomes (Sorkin regulate food intake (Stice et al. 2008; Beaulieu and von Zastrow 2009). Interestingly, adrener- and Gainetdinov 2011). D2DR controls behav- gic stimulation of Erk is reduced in cells with ior associated with food intake and energy impaired b2-adrenergic receptor (b2-AR) en- expenditure (Meguid et al. 2000). D2DR under- docytosis (Daaka et al. 1998). Hence, GPCR goes rapid internalization leading to desensiti- membrane traffic and signaling are intimately zation following stimulation with dopamine or related, with specific signals emanating from other ligands (Gainetdinov et al. 2004), and this distinct cellular locale(s). GPCRs show indi- occurs via clathrin-dependent (Paspalas et al. vidual signatures of GRK-dependent GPCR 2006) or -independent mechanisms (Vickery phosphorylation, b-arrestin binding, and ubiq- and von Zastrow 1999). Agonist-stimulated in- uitylation. Here, we examine the control of ternalization of D2DR is GRK- and b-arrestin- metabolism by endocytosis of b2-AR and D2- independent, and hence involves a noncanon- dopamine receptor (D2DR), which contribute ical endocytic mechanism for receptor desen- to the peripheral and central regulation of me- sitization, although b-arrestins do contribute tabolism, respectively. to D2DR signaling (Namkung et al. 2009). The adaptor protein CIN85 (Shimokawa et al. 2010) as well as the BLOC-1 complex (Iizuka Adrenergic Control of Lipolysis Depends et al. 2007) are required for agonist-stimulated on b2-AR Endocytosis D2DR internalization, and subsequent recycl- Agonist stimulation of b2-AR in adipocytes ing leading to D2DR resensitization occurs causes release of fatty acids from triglyceride via a slow Rab-11-dependent pathway (Li et al. stores. Adrenergic stimulation elicits rapid b2- 2012). Importantly, mice lacking neuronal AR CME. b2-AR then undergoes Rab11-depen- CIN85 that showed defective D2DR endocy- dent recycling to the cell surface (Seachrist et al. tosis in the striatum were lean despite having

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elevated food intake (Shimokawa et al. 2010), mTOR as a branched chain amino acid sensor suggesting that D2DR endocytosis may impact (Tokunaga et al. 2004), suggests the existence of on energy expenditure (or may reflect action of elaborate coordination of nutrient uptake, au- other CIN85-dependent receptors). These re- tophagy, and hormone signaling that we are sults exemplify how endocytosis in neurons crit- only beginning to understand. ically impacts on systemic metabolism. Future work should examine how the met- abolic functions of the many cellular carriers, transporters, and receptors (e.g., lipid, nucleo- CONCLUDING REMARKS AND FUTURE side or amino acid transporters, other signaling DIRECTIONS receptors) are impacted by their endocytosis. Wehave examined how the regulated endocyto- Such information, along with a better under- sis of nutrient carriers (e.g., TfR1, LDLR), standing of the molecular underpinnings, will transporters (GLUTs and Na/K-ATPase), and provide important insight into human metab- signaling receptors (IR, b2-AR, D2DR) facili- olism and may generate novel corrective thera- tate or limit the function of these important pies for metabolic disease. regulators of systemic and cellular metabolism. For many of these examples, the regulation of endocytosis is well documented, and a critical ACKNOWLEDGMENTS physiological role for endocytosis in the ho- C.N.A. is supported by operating grant No. meostasis of various nutrients is emerging. 125854 from the Canadian Institutes of Health Nonetheless, there is still much to learn about Research. A.K. is supported by Operating grant the molecular mechanisms regulating carrier, No. 3707 from the Canadian Institutes of transporter, and receptor endocytosis within Health Research. T.E.M. is supported by NIH the context of metabolic homeostasis. RO1 DK52852. Furthermore, given the diversity of mem- brane proteins at the cell surface involved in the cellular uptake and systemic regulation of REFERENCES metabolites, it is surprising that relatively little Reference is also in this collection. is known about how the endocytosis of most transporters and receptors controls their func- Acconcia F,Sigismund S, Polo S. 2009. Ubiquitin in traffick- tion. Importantly, approximately half of the 590 ing: The network at work. Exp Cell Res 315: 1610–1618. human kinases (protein, lipid, and carbo- Alves DS, Farr GA, Seo-Mayer P, Caplan MJ. 2010. AS160 associates with the Naþ,Kþ-ATPase and mediates the hydrate) are required for the normal endo- adenosine monophosphate-stimulated protein kinase- membrane traffic of vesicular stomatitis virus dependent regulation of pump surface expres- (VSV) and/or Simian virus 40 (SV40), markers sion. Mol Biol Cell 21: 4400–4408. Antonescu CN, Diaz M, Femia G, Planas JV, Klip A. 2008a. of clathrin-dependent and clathrin-indepen- Clathrin-dependent and independent endocytosis of dent endocytosis, respectively (Pelkmans et al. 4 (GLUT4) in myoblasts: Regulation 2005). Indeed some of the kinases found to reg- by mitochondrial uncoupling. Traffic 9: 1173–1190. ulate endomembrane traffic are metabolic en- Antonescu CN, Randhawa VK, Klip A. 2008b. Dissecting GLUT4 traffic components in L6 myocytes by fluores- zymes, such as FN3KRP, which controls protein cence-based, single-cell assays. Meth Mol Biol 457: 367– modification by fructosamine (Pelkmans et al. 378. 2005). This suggests that endomembrane traffic Antonescu CN, Foti M, Sauvonnet N, Klip A. 2009. Ready, is tightly coupled to or controlled by a cell’s set, internalize: Mechanisms and regulation of GLUT4 endocytosis. Biosci Rep 29: 1–11. metabolic state, and that we have only begun Backer JM, Kahn CR, White MF. 1989. Tyrosine phosphor- to understand this regulatory process. Further- ylation of the insulin receptor during insulin-stimulated more, that mTOR was found to exert broad ef- internalization in rat hepatoma cells. J Biol Chem 264: fects on the cell-surface membrane traffic of nu- 1694–1701. Bagrov AY, Shapiro JI, Fedorova OV.2009. Endogenous car- merous transporters and receptors in yeast diotonic steroids: Physiology, pharmacology, and novel (MacGurn et al. 2011), and the central role of therapeutic targets. Pharmacol Rev 61: 9–38.

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20 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016964 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

Reciprocal Regulation of Endocytosis and Metabolism

Costin N. Antonescu, Timothy E. McGraw and Amira Klip

Cold Spring Harb Perspect Biol 2014; doi: 10.1101/cshperspect.a016964

Subject Collection Endocytosis

Endocytosis: Past, Present, and Future Imaging and Modeling the Dynamics of Sandra L. Schmid, Alexander Sorkin and Marino Clathrin-Mediated Endocytosis Zerial Marcel Mettlen and Gaudenz Danuser Rab Proteins and the Compartmentalization of the Endocytic Accessory Factors and Regulation of Endosomal System Clathrin-Mediated Endocytosis Angela Wandinger-Ness and Marino Zerial Christien J. Merrifield and Marko Kaksonen Cargo Sorting in the Endocytic Pathway: A Key The Complex Ultrastructure of the Endolysosomal Regulator of Cell Polarity and Tissue Dynamics System Suzanne Eaton and Fernando Martin-Belmonte Judith Klumperman and Graça Raposo Unconventional Functions for Clathrin, ESCRTs, The Biogenesis of Lysosomes and and Other Endocytic Regulators in the Lysosome-Related Organelles Cytoskeleton, Cell Cycle, Nucleus, and Beyond: J. Paul Luzio, Yvonne Hackmann, Nele M.G. Links to Human Disease Dieckmann, et al. Frances M. Brodsky, R. Thomas Sosa, Joel A. Ybe, et al. Endocytosis of Viruses and Bacteria Endocytosis, Signaling, and Beyond Pascale Cossart and Ari Helenius Pier Paolo Di Fiore and Mark von Zastrow Lysosomal Adaptation: How the Lysosome Clathrin-Independent Pathways of Endocytosis Responds to External Cues Satyajit Mayor, Robert G. Parton and Julie G. Carmine Settembre and Andrea Ballabio Donaldson Reciprocal Regulation of Endocytosis and The Role of Endocytosis during Morphogenetic Metabolism Signaling Costin N. Antonescu, Timothy E. McGraw and Marcos Gonzalez-Gaitan and Frank Jülicher Amira Klip Endocytosis and Autophagy: Exploitation or Role of Endosomes and Lysosomes in Human Cooperation? Disease Sharon A. Tooze, Adi Abada and Zvulun Elazar Frederick R. Maxfield

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Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

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Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved