In 2007 the G. L. Brown Prize Lecture was given by Professor C. A. R. Boyd at the Universities of Dundee, Belfast, London (St George's), Warwick, Liverpool, Cambridge and Oxford.
Corresponding author C. A. R. Boyd: Department of Physiology, Anatomy & Genetics, University of Oxford, Le Gros Clark Building, South Parks Road, Oxford OX1 3QX, UK. Email: firstname.lastname@example.org
The hallmark of epithelial cells is their functional polarization. It is those membrane proteins that are distributed differentially, either to the apical or to the basal surface, that determine epithelial physiology. Such proteins will include ‘pumps’, ‘channels’ and ‘carriers’, and it is the functional interplay between the actions of these molecules that allows the specific properties of the epithelium to emerge. Epithelial properties will additionally depend on: (a) the extent to which there may be a route between adjacent cells (the ‘paracellular’ route); and (b) the folding of the epithelium (as, for example, in the loop of Henle). As for other transporters, there is polarized distribution of amino-acid carriers; the molecular basis of these is of considerable current interest with regard to function, including ‘inborn errors’ (amino-acidurias); some of these transporters have additional functions, such as in the regulation of cell fusion, in modulating cell adherence and in activating intracellular signalling pathways. Collaboration of physiologists with fly geneticists has generated new insights into epithelial function. One example is the finding that certain amino-acid transporters may act as ‘transceptors’ and play a role as sensors of the extracellular environment that then regulate intracellular pathways controlling cell growth.
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The purpose of the G. L. Brown Lecture is ‘to stimulate an interest in Physiology’. For me, this is a challenge I accept with pleasure since throughout my professional life I have both enjoyed being a physiologist professionally and similarly have enjoyed teaching physiology as a discipline. The individual after whom this lecture is named, G. L. Brown, was in fact one of my own teachers, remembered as a figure with an earthy sense of (Northern) humour; I remember him describing the relationship between stimulus and response in excitable tissues as being like ‘flushing the lavatory, either it flushes or it doesn't’ (this was the ‘all-or-none principle’). By the time Brown was teaching my generation of medical students in Oxford I suspect that the best of his own research was behind him. I was interested in the tale that I was told subsequently, that Brown's enthusiasm for his own experimental science had waned when it became apparent that a phenomenon he and colleagues had described (Brown & Holmes, 1956), post-tetanic potentiation (PTP), was to do more with (artifactual) accumulation of extracellular potassium ions than it was an insight into a fundamental property of the nervous system. [It is interesting to realize that this work preceded the studies of Bliss & Lomo (1973) on long-term potentiation by some 15 years, both groups having worked at the same institution in Mill Hill.]
D. S. Parsons stimulated my own interest in epithelial physiology, and in the year of his ninetieth birthday it is particularly nice to be able to acknowledge his nurturing of this discipline in the UK. It is, however, the very widely acknowledged insight of a Scandinavian physiologist that I wish to mention briefly by way of introduction.
H. H. Ussing (1905–2000), together with Koefoed-Johnsen, published a key paper some 50 years ago entitled ‘The nature of the frog skin potential’ (Koefoed-Johnsen & Ussing, 1958). This paper for the first time sought to explain the fundamental property of epithelial cells, namely their ability to achieve net transepithelial transport, by the asymmetric distribution between the two faces of the cell of ‘pumps’ and of ‘conductances’ (see Fig. 1A). Out of this came the key insight of how, in every epithelium, transepithelial transport, whether absorptive or secretory, emerges from asymmetric distribution of pumps, channels and carriers (see Fig. 1B).
My own personal interest arose from work on non-electrolyte transport in epithelia. This field had been developed by a number of scientists (including Parsons himself, e.g. Fisher & Parsons, 1953) in the 1950s but the insight from this time that remains in all current text books is the notion of Robert Crane published originally as an appendix to a symposium paper published in 1960 (Crane et al. 1960). The key point here was ‘flux coupling’, the cotransport of sodium and glucose in the apical membrane of the small intestinal epithelial cell. Half a century later this idea has turned into one of the most studied of all transporter proteins (SGLT1), the sodium–glucose cotransporter. Although not appreciated until later, it was the coupling of an ionic flux (Na+) to the movement of an uncharged solute (glucose) that connected the ‘electrical’ insights of Ussing to the field of non-electrolyte transport, and it is this that I will develop in this lecture.
Observations on amino acid transport in small intestine
In the 1970s, working in Oxford, D. S. Parsons, C. I. Cheeseman and I developed a vascularly perfused preparation of small intestine (Boyd et al. 1975); part of the purpose of this was to overcome the issue of artifactual accumulation of solute (analogous to the artifact mentioned above for PTP). We felt that in this way we would be able to find out something of the properties of the ‘dark side’ of the epithelium, the blood-facing basolateral membrane. At this time little was known of transport events for any solute in this membrane. An innovation that Parsons and I developed subsequently (Boyd & Parsons, 1979) was to use non-steady-state perturbation; that is to say, we loaded the epithelial cells with (radiolabelled) sugar or amino acid, and subsequently during the ‘washout’ studied quantitivity the impact of altering conditions in either the lumen or the blood. This approach proved useful in glucose transport, for example in distinguishing the chemical specificity for substrate recognition in the basal and apical membrane, and provided a basis for studies with vesicles that were reported very soon after. It was also gratifying that this technique was used subsequently to study transport in quite different epithelia (e.g. Mann et al. 1989), work that ran in parallel with studies on capillary endothelial physiology (e.g. Crone, 1973; Yudilevich & Sépulveda, 1976).
One particular set of observations that intrigued me was made subsequently in Canada by Cheeseman using this preparation (Cheeseman, 1983; Fig. 2). He reinvestigated a phenomenon described earlier by Munck & Schultz (1969) and by Reiser & Christiansen (1971); both sets of authors had noted when observing the accumulation of the cationic amino acid lysine in epithelial cells or tissue in vitro that, unexpectedly, addition of the (chemically quite distinct) neutral amino acid leucine (uncharged at physiological pH) diminished intracellular lysine accumulation. One suggestion was that this interaction might not be occurring at the apical (brush-border) membrane. Cheeseman's data showed that this indeed was the case and indicated that, through an ill-defined interaction at the basal membrane, extremely low (micromolar) concentrations of leucine allowed lysine to leave the cell. These data led me to postulate a model for cationic amino-acid transport across epithelia (Fig. 3A); the basic features of this model still apply today (Fig. 3B). The model was based on the identification of a novel amino-acid transport system, y+L.
Discovery of the amino-acid transport system y+L
R. Devés discovered transport system y+L in human red blood cells whilst working in Oxford (Devés et al. 1992). It was identified as a novel transport system for the amino acid lysine that was inhibited with very high infinity by leucine. In the original description, it was noted that the transport system y+L was not the only mechanism for lysine entry, the previously described system y+ also contributing to the total flux. Successful functional separation of the two systems depended on kinetic analysis, requiring development of some novel insights into the mathematical distinction between parallel transport systems with overlapping specificities for substrates (lysine and leucine) but differing affinities for their interactions with two independent transport processes (Fig. 4). The Devés et al. (1992) paper represents one of the strengths that physiology brings to biology, namely a willingness to follow through mathematical modelling and quantitative analysis to allow prediction and experimental test of predictions. Soon after the original description of system y+L, two additional features emerged. The first was technical, but of particular value experimentally; the sulphydryl reagent NEM did not react with y+L but did with system y+, which was inactivated by chemical modification, thus allowing the properties of y+L to be analysed rigorously in an independent manner (Devés et al. 1993). The second was a physiological observation, the finding that the interaction of the amino acids lysine and leucine differed spectacularly with respect to sodium dependence (see Fig. 4). For leucine to interact with the y+L transporter, sodium (as opposed to a sodium-free medium) increased binding by a factor of 300 but for lysine no such effect occurred (Angelo & Devés, 1994). This feature makes this transport system of particular interest to a cellular physiologist, given the (relatively) high extracellular and (relatively) low intracellular concentrations of sodium ions. Additional features of system y+L included the strong exchange properties of the transporter and the insensitivity of the translocation process to membrane potential (Fig. 5). Moreover, it was realized very quickly that this novel system was not confined to erythrocytes but was also observed, for example, in T lymphocytes (Boyd & Crawford, 1992) and in epithelial trophoblast cell membranes (Eleno et al. 1994). It was this combination of properties that led to the proposal that system y+L might underpin the movement of cationic amino acids across the basolateral membrane of epithelial cells, for example in intestine, in kidney and elsewhere (e.g. in lung), as proposed in Fig. 3A.
Lysinuric protein intolerance and amino-acid transport disorders
Soon after the discovery of system y+L and the suggested central role that it plays in epithelial amino-acid transport, a genetic knockout confirmed the proposal. The knockout, however, was not in a model organism but in humans, and had not been engineered but was a classical Mendelian monogeneic disease. The key observations were made by Finnish medical scientists working on the exceptionally rare monogeneic recessive disorder responsible for the (largely paediatric) disease, lysinuric protein intolerance (LPI; Norio et al. 1971). This disease causes devastating metabolic abnormalities, probably associated with urea cycle abnormalities secondary to arginine deficiency. Both cationic amino acids, lysine and arginine, are malabsorbed from the intestine and similarly are not reabsorbed in the nephron, leading to a characteristic amino-aciduria that originally allowed identification of such individuals. In the late 1970s, J. Desjeux and colleagues (Desjeux et al. 1980) had already shown that this disorder had as its functional locus abnormal lysine transport at the basal membrane of intestinal biopsies, and this elegant work fitted with the demonstration (Rajantie et al. 1980) that in vivo testing of absorption was only compatible with a defect in the basal membrane, since in LPI patients following oral ingestion there was no increment in plasma lysine concentrations whether the amino acid was given as part of a dipeptide (Gly–Lys) or as a free amino acid (Lys). The logic behind this in vivo experiment was instructive, it being known that peptide transporters, found exclusively in the apical membrane, would circumvent any defect in apical amino transport of amino acid, but would not be able to do so for defects in amino-acid transport in the basal membrane. Hence the discovery (Torrents et al. 1999; Borsani et al. 1999) that the gene that was mutated in LPI was indeed the gene that encoded (part of) the y+L transporter, and that this gene product was functionally located in the basal membrane of epithelia thus confirmed the 1992 model (Fig. 3A) regarding the role in epithelial cells of the system y+L basolateral membrane transporter. Very recently (Sperandeo et al. 2007), the properties of a mouse knockout for the y+LAT1 gene have been described. This mouse showed some of the pathology predicted (in particular, neurological defects when fed a protein-rich diet apparently caused by acute hyperammonaemia). Additionally, the homozygous animals were small at birth, and this intra-uterine growth restriction appeared to result from reduced expression of insulin-like growth factor (Igf) 1 and 2, growth factors of particular importance in early development. Perhaps the most surprising finding (from microarray analysis of these animals) was the marked downregulation of the intestinal Na+–Pi cotransporter. The mechanism of this effect is not yet clear; I speculate that it may result from raised intracellular concentration of Pi. This might result from inadequate rates of Pi incorporation into ATP, since synthesis of such high-energy molecules is compromised by decreased glutamine entry through system y+L into the enterocytes from the blood, glutamine (rather than glucose) being the major metabolic fuel for both enterocytes and for lymphocytes (Ardawi & Newsholme, 1990).
Very intriguingly, system y+L is now known to be a transporter that is heterodimeric, that is, it is made up of two distinct subunits (see, e.g. Palacín et al. 2005). Moreover, the heterodimeric structure (see Fig. 6) is held together by a covalent extracellular bond between a ‘light chain’ and a ‘heavy chain’. The heavy chain is now known as CD98 (previously 4F2) and had been identified in the early 1980s by immunologists seeking to identify surface molecules on activated lymphocytes (Haynes et al. 1981). The story does not stop with system y+L, however, since CD98 is also known to make similar covalent structures with no less than six other light chains, and in each case the specific heterodimer encodes a functioning (different) amino-acid transport system (see Fig. 3B for another example, the system L amino acid transporter). Functionally, all of these transporters appear to work as exchangers (and so do not generate net amino-acid movement, Ramadan et al. 2007). They differ in their substrate specificity and in their cellular distribution, but in epithelial cells they are all confined to the basal membrane; indeed, the heavy chain, CD98, has been used as an experimental marker for basal membrane identification (Liu et al. 2003).
CD98 is the heavy chain for this particular subfamily of amino-acid transporters; its ‘opposite number’ as a heavy chain confined to the apical membrane of epithelial cells is known as rBAT (‘related to broad system amino-acid transporter’). The heavy chain rBAT forms heterodimers with a distinct separate group of light chains that in intestine and in the kidney form the basis for entry of specific groups of amino acids into the cell across the apical membrane (see again Fig. 3B). One group of amino acids transported in this way are the relatively insoluble sulphur-containing amino acids, cysteine and the cross-linked cystine. Owing to their low solubility, when renal reabsorption is impaired they tend to precipitate as water continues to be reabsorbed from the nephron more distally, giving rise to the (relatively) common disease cystinuria, one of the four original ‘inborn errors of metabolism’ described by Garrod (1906). Rather beautifully, it was recognized by careful clinical study that two specific subtypes of the disease could be detected phenotypically (Palacín et al. 2005). The relationship between these two groups (silent heterozygotes and hyperexcreting heterozygotes) and the relevant heavy and light chains for this transporter are the subject of on-going interest (Palacín et al. 2005).
Comparison of the two diseases, LPI and cystinuria, is instructive; in the context of the Ussing model they show that two particular amino-acid carriers have polarized distribution to, respectively, the basal and apical membranes of the intestinal and renal epithelium. Thus we now have a handle on the signals that determine to which membrane these specific light chains of the transporters are distributed; and those signals are encoded within the structures of the respective heavy chains, CD98 and rBAT.
Other functions of CD98
CD98 is a membrane protein with a single transmembrane domain (Devés & Boyd, 2000). It has a short amino-terminal sequence that is cytoplasmic, and a very large, heavily glycosylated extracellular carboxy-terminus. As a result of its disulphide covalent attachment to individual members of the light chain family, its molecular weight under normal (non-reduced) conditions is approximately 120 kDa; however, when SDS-PAGE is carried out under reducing conditions, breaking the intermolecular disulphide bond, two bands are found: CD98 itself, running with a molecular weight of approximately 80 kDa, and the light chain(s), running with a molecular weight of 40 kDa. CD98 was found, when sequenced, to be identical to the previously identified protein FRP-1 (fusion regulatory protein 1). This already hinted at the fact that CD98 has functions other than its role in trafficking (Nakamura et al. 1999) amino-acid transporter light chains to the correct plasma membrane.
Recent work shows that CD98, expressed in many different tissues (notably those that are actively proliferating), has a specific role in cell adhesion. This function emerged from studies designed to detect proteins that regulated integrin function using an in vitro genetic screen. Fenczik et al. (1997) showed that, uniquely, CD98 was able to activate α3 and β1 integrins, presumably via cytoplasmic interactions between the short intracellular tail of CD98 and the intracellular portion of these two integrin proteins. Given the role of integrins in epithelial cell adherence to the underlying basal lamina (basement membrane), this is a particularly interesting observation, and one that may relate to recent interest in the role of CD98 in epithelial tumour (carcinoma) metastasis. It is obviously important for future work on the biology of carcinoma that loss of adherence of epithelial cells is better understood, and CD98 is an interesting candidate molecule in this regard.
Another novel feature of CD98 relates to its specific roles in the immunology of T-cell activation. There has recently been renewed interest in T-cell amino-acid metabolism and naturally this extends to understanding the relevance to this process of the amino-acid transporters they express. For example, the amino acid glutamine is a substrate for system y+L amino-acid transporters. It seems at least plausible that one of the reasons why CD98 is upregulated in activated T-cells relates to this particular role in T-cell nutrition. Very recent interest (e.g. Yeramian et al. 2006) in arginine delivery in the context of T-cell biology may similarly relate to this role of CD98. It is also interesting that specific immunopathology is frequently found in patients with LPI.
Receptor signalling is also implicated in CD98 biology, since the large extracellular domain of CD98 has strong sequence homology with (bacterial) ‘amylase-like’ domains. Earlier work had raised the possibility of galectin binding (Dong & Hughes, 1997), and recently Dalton et al. (2007) have shown, by co-immunoprecipitation of CD98 and of galectin 3, that there is indeed a direct molecular interaction between the two proteins. This may relate to a very particular function of CD98, namely its role in cell fusion. Although often considered ‘pathophysiological’, there are at least three ‘normal’ circumstances in which cell fusion occurs in mammals. An obvious one is the fusion of sperm and egg at fertilization; formation of osteoclasts in normal bone remodelling is another; a third is myocyte fusion in skeletal muscle development. Here I will describe evidence that CD98 plays a central role in the fusion of an epithelium, the trophoblast cells of the placenta. In human pregnancy, this fused epithelium (the syncytiotrophoblast) forms the cellular boundary separating the maternal circulation (in direct contact with the apical surface of the trophoblast) from fetal blood flowing in capillaries immediately deep to the basal surface of the epithelium. Fusion of the trophoblast is thus central to the success of viviparity. The syncytiotrophoblast is involved not only with gas exchange, nutrient delivery, waste product removal, hormone biosynthesis and metabolism but also in controlling the immune interface between mother and baby. The trophoblast thus is formed by fusion of the individual cellular elements (cytotrophoblasts) to become a polarized epithelium with a different set of pumps, channels and carriers in the apical (brush-border) from those in the basal membrane.
Figure 7 (from Kudo & Boyd, 2004) shows the results of experiments using single-stranded interfering-RNA (siRNA) to test the possible role of CD98 in the process of placental fusion. The siRNA molecules used were designed to inhibit expression of CD98 protein (as verified by Western blotting), in contrast to the scrambled (control) siRNA sequences. A novel assay for cell fusion was developed using flow cytometry. Fusion was measured following the mixing of populations of cells that expressed either a green fluorescent protein (GFP)-tagged histone or a red-shifted GFP-tagged cytochrome oxidase. The number of double-labelled cells was then determined (Kudo et al. 2003). Trophoblast cells can be induced to fuse by agents such as forskolin that raise cAMP concentrations in the cells. Figure 7 shows clearly that the number of fused cells is indeed increased dramatically by this experimental procedure, and that under such conditions knockdown of CD98 by siRNA transfection decreases fusion markedly. The possibility that galectin 3 is involved in this, given that it is a bivalent molecule with two binding sites, is obviously attractive. For example, it is possible to predict that a bivalent galectin 3 molecular structure could bind and cross-link two adjacent CD98-expressing cells, and in this way initiate (possibly via integrin activation) the downstream processes necessary for trophoblast synsytialization. Whether this mechanism applies to other examples of cell fusion in biology is at present an open question.
A comparative view of epithelial amino-acid transporters: the role of the proton-assisted transporter family (PAT) in cell signalling
In an attempt to investigate the role of additional functions of heterodimeric amino-acid transporters in an in vivo context, a collaborative programme with Drosophila biologists was undertaken, the fruit fly having great genetic tractability. Physiologically, these studies showed that manipulation of transporter expression (overexpression, underexpression either global or tissue specific) influenced biological processes of cell growth in unexpected ways, implying that some amino-acid transporters have unusual roles in cell signalling. Here I will focus on the discovery that certain members of the proton-assisted transporter family (the PATs) play such roles in the fruit fly. Goberdhan et al. (2005) and Wilson et al. (2007) provide the context for these experiments, which showed that one member of the PAT family (which was named ‘path’) was unusual in that functionally it was an exceptionally low-capacity/high-affinity transporter yet it was able to strongly influence insulin signalling pathways affecting cell growth. An interpretation (Fig. 8; Reynolds et al. 2007) is that these types of transporters must, by detecting the extracellular amino-acid environment, regulate intracellular cascades of signalling molecules (e.g. at the level of the kinase mammalian target of rapamycin (mTOR)) that themselves regulate transcriptional machinery. Such molecules have been described in yeast and are sometimes referred to as ‘transceptors’ because of their hybrid function, sitting between transporters and receptors. It is particularly interesting that Hyde et al. (2007) very recently have described analogous properties in mammalian cell lines. This field is clearly a fertile one for further experimental work, given the lack of knowledge of the specific functions of individual members of gene families encoding different transporters. In the mammalian context, one hypothesis is that PATs 3 and 4 might act as transceptors, analogous to path; if indeed this is the case, they become interesting targets for manipulation in diseases where cell growth is dysregulated (see Fig. 8). The combination of cell physiology (looking at transport mechanisms and identifying systems by quantitative kinetic analysis), of cell expression studies (using Xenopus oocyte expression with functional assays carried out through electrophysiology and flux studies), coupled with in vivo work on a model organism (with well-characterized genetics), makes amino-acid transport a lively and experimentally challenging field with implications across many areas of physiology, including development, endocrinology, immunology and the nervous system. Ussing should continue to be recognized and applauded for the original insights he had; one hopes that he would also be pleased to see the harvest that continues to be reaped from the fields whose seeds he planted.
I would like to acknowledge many colleagues from all parts of the world with whom I have had the great privilege and pleasure of working. I am also particularly grateful to Bob Laynes for technical support, and to funding bodies (Wellcome Trust, Cancer Research) UK) for the financial support of my laboratory.