Single proteins might have dual but related functions in intracellular and extracellular microenvironments

Abstract

The maintenance of organ homeostasis and the control of an appropriate response to environmental alterations require the intimate coordination of cellular functions and tissue organization. An important component of this coordination could be provided by proteins that can have distinct but linked functions on both sides of the plasma membrane. We present a model that proposes that unconventional secretion provides a mechanism through which single proteins can integrate complex tissue functions.

Single genes can exert complex and dynamic effects through a number of different processes that multiply the functions of gene products. Alternative splicing can create many different transcripts from a single gene, which can encode proteins that have diverse, even antagonistic, functions. Post-translational modifications can alter the stability, activity, localization and even the basic functions of proteins. Furthermore, a protein can exist in different subcellular locations. More recently, it has become clear that single proteins can function both inside and outside of the cell. These proteins often lack defined secretory signal sequences and transit the plasma membrane by mechanisms that are distinct from the classical endoplasmic reticulum (ER)–Golgi secretory process.

When examples of such proteins are examined individually, the multifunctionality of these proteins and the lack of a signal sequence are puzzling — why should a protein with a well-known function in one context function in such a distinct way in another? We propose that one reason for this is so that the protein can coordinate the organization and maintenance of a global tissue function. Here, we describe three examples of proteins that have intracellular and extracellular roles, we outline their specific functions in the extracellular and the intracellular space, and we discuss how these functions might be linked. All of these proteins have been reported to transit the plasma membrane through unconventional secretory mechanisms. Therefore, we also discuss the possible relationship between unconventional secretion and the coordination of extracellular and intracellular events, and how this relationship might be used to identify other proteins that share these characteristics.

Coordinating tissue organization

In this section, we discuss epimorphin and syntaxin 2, amphoterin and high mobility group protein B1 (HMGB1), and tissue transglutaminase (TTG; also known as TGM2). Syntaxin 2 might coordinate the morphogenesis of secretory organs (a role that was originally attributed to epimorphin, which is encoded by the same gene) with the control of protein secretion; HMGB1 might link inflammation (a role that was originally attributed to amphoterin, which is encoded by the same gene) with the regulation of gene expression; and TTG affects the delivery of and response to apoptotic signals on both sides of the plasma membrane.

Morphogenesis and protein secretion

Epimorphin was initially identified as the target of a monoclonal antibody that blocked hair follicle morphogenesis in the dermal epithelium¹, but it is now known to be involved in the morphogenesis and development of many other epithelial organs (FIG. 1a; reviewed in REF. 2). In the mammary gland, epimorphin directs branching and luminal morphogenesis, and the orientation of its presentation to the mammary epithelium dictates the resulting tissue structure. The presentation of epimorphin to cells in a polarized fashion stimu lates branching morphogenesis pro cesses that are associated with increasing the complexity of the mammary ductal epithelium, whereas apolar presentation stimulates epithelial structures to increase the size of their ductal lumen³. Inappropriate expression of epimorphin in transgenic animals can lead to the develop ment of enlarged and cystic ducts, cell proliferation and progression to cancer in aged animals⁴.

Figure 1. Models of linked intracellular and extracellular roles for molecules of dual topology or multiple function.

Outside the cell, epimorphin mediates tissue morphogenesis, whereas inside the cell, syntaxin 2 (which is encoded by the same gene but might differ in structural modifications) controls protein secretion from endoplasmic reticulum–Golgi-derived vesicles (a,b). Outside the cell, amphoterin stimulates cell responses that are mediated by altered gene expression, whereas inside the cell, high mobility group protein B1 (HMGB1; which is encoded by the same gene but might differ in structural modifications) controls chromatin organization and influences the expression of many gene products (c,d). Outside the cell, tissue transglutaminase (TTG; also known as TGM2) controls extracellular matrix (ECM) organization, whereas inside the cell, it participates in pathways that determine cell survival or apoptosis and are responsive in part to cell attachment to the ECM (e,f).

Epimorphin and syntaxin 2 are encoded by the same gene and are likely to be the same protein (REF. ⁵). However, although untested, the proteins might have different structural modifications that affect their localization and functions. Syntaxins are members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) super-family of membrane proteins that mediate intracellular vesicle docking and fusion⁶. SNARE proteins are crucial during the final stage of membrane fusion, which depends on the formation of a protein complex that includes vesicle-associated membrane protein (VAMP) on the vesicle surface, and 25 kDa synaptosome-associated protein (SNAP25) and a syntaxin on the target surface. When assembled into a functional complex, these three molecules form an intermolecular four-helix bundle that initiates the membrane fusion process. There are many distinct mammalian syntaxins, each associated with a particular vesicle fusion process. Syntaxin 2 was discovered soon after the initial identification of epimorphin⁷. It is a plasma-membrane-associated protein, but its functional domain is orientated to the inside of the cell (FIG. 1b). Syntaxin 2 participates in specific and crucial membrane fusion processes, including granule release from platelets⁸, the secretion of lung surfactant from alveolar type II cells⁹ and the regulation of midbody abscission during cytokinesis¹⁰.

Most of the proteins that are found outside of the cell are trafficked through the ER–Golgi pathway. These proteins are initially targeted to the ER by the presence of a leader sequence, a hydrophobic signal peptide that is usually located at the amino terminus of the protein (BOX 1). The discovery that the same gene encodes both epi morphin and syntaxin 2 provoked substantial controversy^11,12, as these proteins lack a leader sequence that would target them to the extracellular space and because there was no known mechanism for their secretion at that time. For many years thereafter, studies of these proteins focused either on their extracellular or intra cellular functions², and the question of how the lead er less syntaxin 2 crosses the plasma membrane came to eclipse the question of why a single protein might have such different roles.

Box 1. Conventional and unconventional protein secretion.

Several different mechanisms have been identified by which cytosolic proteins can cross the plasma membrane.

Conventional secretion

During polypeptide assembly by the ribosome, the presence of a membrane-targeting signal sequence leads to translocation of the ribosome–polypeptide complex to the endoplasmic reticulum (ER) by the signal recognition complex (see the figure, part A, step 1). Following validation of correct protein folding in the ER, proteins are captured in transitional vesicles that bud from the ER and translocate to and fuse with the Golgi (see the figure, part A, step 2). Protein maturation in the Golgi involves post-translational modifications, including the attachment of sugar molecules that can significantly alter the surface structure and function of the protein (see the figure, part A, step 3). Once fully processed, proteins are transported to the cell surface through targeted vesicles (see the figure, part A, step 4).

Unconventional secretion

Proteins that lack signal sequences can directly transit the plasma membrane by coordinating with specialized cotransport complexes (see the figure, part Ba), as is the case for fibroblast growth factor 1 (FGF1) and FGF2. Some proteins can be collected into secretory lysosomes or vesicles that fuse with the plasma membrane and release their contents into the extracellular space (see the figure, part Bb). This mechanism has been shown for amphoterin and high mobility group protein B1 (HMGB1; which are encoded by the same gene but might differ in structural modifications) and interleukin 1β (IL-1β). An alternative method for unconventional secretion involves membrane blebbing to generate exovesicles (see the figure, part Bc). These are lysed to release their contents into the extracellular space. It should be noted that these are only a subset of mechanisms that are implicated for unconventional secretion. Other mechanisms include secretory lysosomes and vesicle shedding (for a more complete discussion of the current understanding of unconventional secretion mechanisms, see REFS ^31,47,48).

We propose that the activity of extracellular epimorphin as a morphogen of epithelial tissues and the activity of intra cellular syntaxin 2 as a mediator of the fusion of secretory vesicles with the plasma membrane might both contribute directly to tissue organization. Secretory epithelial organs depend on correct morphogenesis (to define sidedness of secretion) and on the correct orientation of secretion, and therefore the functions that are fulfilled by epimorphin/syntaxin 2 are complementary. Given the potential importance of proper regulation of this protein in normal tissue function, it should not be surprising that overexpression and misregulation of epimorphin/syntaxin 2 can cause organ pathology and the induction of cancer^3,4.

Cell communication and gene expression

Whereas epimorphin/syntaxin 2 coordin ates the maintenance and development of normal tissue structures, amphoterin/HMGB1 might coordinate the responses of tissues that are damaged or are under going inflammatory processes (reviewed in REFS ^13-15). Amphoterin is an extracellular protein (FIG. 1c) that was first identified in the brain, in which it was shown to mediate outgrowth of neurites. It has since been implicated as an inducer of chemotaxis and cell motility, and as a crucial cytokine that mediates inflammatory responses. Amphoterin stimulates motility and inflammation through binding to specific extracellular receptors, including receptor for advanced glycosylation end products (RAGE; also known as AGER). The interaction of RAGE and amphoterin is of particular interest because these two molecules have also been shown to regulate the invasiveness of several tumour cell types, providing insight into potential new therapeutic opportunities in the treatment of cancer. Amphoterin also lacks a classical secretion sequence, and although it can be released from necrotic cells, it can also be secreted from intact cells in response to specific stimuli, including the cytokines tumour necrosis factor (TNF) and interleukin 1β (IL-1β)¹⁶ or the extracellular matrix (ECM) protein laminin¹⁷. Studies from monocytes have indicated that a vesicle-mediated secretory pathway mediates the unconventional secretion of amphoterin (REF. ¹⁸) (BOX 1).

HMGB-family proteins are non-histone components of chromatin. HMGB1 was one of the first members of this family to be discovered. HMGB proteins function primarily as architectural facilitators in the assembly of nucleoprotein complexes^19,20. HMGB1 is ubiquitously expressed and is localized to the nucleus in most cell types (FIG. 1d). Although HMGB1 can bind with low affinity to single-stranded, linear duplex and supercoiled DNA, it binds with high affinity to cruciform DNA, a form of DNA that can be produced as an intermediate in chromosomal recombination²¹. The association of HMGB1 with DNA leads to the assembly of nucleoprotein complexes that affect and coordinate gene transcription^19,20,22. Amphoterin/HMGB1 thus functions in the nucleus as a modulator of chromatin function and gene expression, and outside the cell as an inducer of chemotaxis, cell motility and inflammation. These cellular behaviours all depend on the coordinated modulation of gene expression.

ECM and cell survival

TTG is found in the nucleus, the cytoplasm, the membrane and the extracellular space, and has been principally characterized as a Ca²⁺- and GTP-regulated protein-crosslinking agent²³ (FIG. 1e,f). Inside the cell, TTG is implicated in an array of physiological processes, most notably the ability to regulate and to induce apoptosis. In the nucleus, TTG transamidates retino blastoma protein (RB), a pro cess that results in the covalent crosslinking of γ-carboxamide groups of Gln residues. This modification of RB provides protection against apoptotic stimuli²⁴. However, when localized to the cytoplasm, TTG can induce apoptosis^24,25. TTG is unique among transglutaminases in that it also possesses GTPase activity and can serve as a signal-transducing G protein²⁶.

TTG lacks a secretory signal sequence but can be exported from the cell by unconventional secretion mechanisms²³. A number of ECM proteins are glutaminyl substrates for TTG, and these proteins, when crosslinked, become resistant to proteolytic degradation and mechanical damage, thereby conferring increased structural stability and flexibility on the tissue architecture. Importantly, degradation of TTG by matrix metallo proteinases facilitates tumour cell invasion and metastasis²⁷. At the cell surface, extracellular TTG facilitates cell–ECM interactions by cross linking the ECM proteins laminin and nidogen²⁸, and also enhances integrin–ECM interactions through transglutamination-independent processes²⁹. The crosslinking function of TTG also has important roles in temporally controlled, tightly regulated processes, such as fibrin clot formation³⁰.

Attachment to the ECM provides important cell survival signals. Non-transformed cells that become detached from the ECM undergo a specific form of apoptosis that is known as anoikis. When localized in the extracellular environment, TTG coordinates extracellular structures and intracellular responses by modulating ECM organization and controls the apoptotic response inside the cell. These functions have been studied primarily as distinct phenomena, but a comparison of the functional roles of TTG in both compartments provides insight into how the processes might be directly connected with each other.

Identifying coordinating proteins

Although epimorphin/syntaxin 2, amphoterin/HMGB1 and TTG are among the best-studied examples of proteins that function on both sides of the plasma membrane, that are secreted by unconventional mechanisms and might function to link inside and outside signalling processes, many others have been investigated (TABLE 1). Receptor for hyaluronan-mediated motility (RHAMM; also known as CD168 and HMMR) coordinates proliferation and mitosis through its roles as a cell surface hyaluronan receptor and its association with mitotic spindles and mitotic signalling pathways³¹. Annexin II (also known as annexin A2) modulates cell–cell and cell–ECM interactions outside the cell³², and modulates cyto kinesis and vesicle trafficking inside the cell^33,34. Extracellular autocrine motility factor (AMF) is a potent cytokine and morphogen^35,36, whereas the identical protein inside the cell is known as cytoplasmic phosphohexose isomerase (PHI; also known as glucose-6-phosphate isomerase (GPI) and phosphoglucose isomerase (PGI)) and controls glycolysis³⁷. Ku controls cell adhesion outside the cell³⁸ and DNA repair in the nucleus³⁹. Although there are considerable differences between the sequences, structures and functions of these proteins, several common features can be observed, including separate structural motifs for the distinct functions of the proteins and the use of unconventional secretion for crossing the plasma membrane.

Table 1.

Molecules with distinct intracellular and extracellular functions

Protein names	Intracellular function	Extracellular function
Epimorphin/syntaxin 2	Vesicle trafficking9 and cytokinesis10	Morphogen2
Amphoterin/HMGB1	DNA-binding protein21	Cytokine14
TTG (also known as TGM2)	Cell signalling24,26	ECM-modifying protein29
RHAMM (also known as CD168 and HMMR)	ERK1 and ERK2 signalling61	Cell surface hyaluronan receptor62
Annexin II (also known as annexin A2)	Cytokinesis and vesicle trafficking33,34	Cell surface receptor32
AMF/PHI (also known as GPI and PGI)	Glycolysis or homeostasis37	cytokine or morphogen35,36
Thioredoxin (also known as ADF)	Redox reactions63	Immunomodulatory cytokine64
Ku	DNA repair39	Cell adhesion38

ADF, adult T-cell leukaemia-derived factor; AMF, autocrine motility factor; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; GPI, glucose-6-phosphate isomerase; HMGB1, high mobility group protein B1; PGI, phosphoglucose isomerase; PHI, phosphohexose isomerase; RHAMM, receptor for hyaluronan-mediated motility; TTG, tissue transglutaminase.

Different motifs for distinct functions

One striking feature of these multifunctional proteins is that the different mechanisms of action can be separated into distinct protein motifs. This is particularly evident with epimorphin/syntaxin 2 (FIG. 2). The SNARE motif is a carboxy-terminal helical domain that is highly conserved in the syntaxin family and is essential for the formation of competent membrane-fusion complexes⁶. The active site of epimorphin for epithelial morpho-genesis has been mapped to an independently folded N-terminal domain that has a three-helix bundle structure⁴⁰. Similarly, the acidic C-terminal domain of HMGB1 seems to be crucial for the regulation of DNA binding and for the formation of ternary structures⁴¹. A specific DDDDE motif in the C terminus of HMGB1 is required for transcription stimulation⁴². By contrast, the extracellular function of amphoterin is dependent on a distinct receptor binding motif that encompasses amino acids 150–183 (REF. ⁴³).

Figure 2. Distinct motifs mediate the different functions of epimorphin and syntaxin 2.

a | A ribbon drawing of the closed conformation of syntaxin 1A⁶⁰ (STX1A) in complex with SEC1 (grey). Molecular coordinates were obtained from the Protein Data Bank, code 1DN1. Syntaxins are composed of three α-helices (blue, green and red) that are connected by a linker sequence (yellow) to the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) helix (white). b | The distinct locations of morphogenic and membrane fusion functional domains. Deletion analysis of epimorphin (EPIM) and STX2 (which are encoded by the same gene but might differ in structural modifications) has shown that mutant molecules that have helices A–C are sufficient to mediate morphogenic activity and that the SNARE domain is dispensible for this⁴⁰, whereas the SNARE domain is specifically required for membrane fusion activity⁶. c | His246 of EPIM and STX2 is the key residue that determines extracellular secretion⁴⁶. Sequence alignment of EPIM/STX2 from mice (m), humans (h), quails (q) and sheep (s) reveals that His246 is conserved, whereas the corresponding position in nonsecreted syntaxins is occupied by an Arg in STX1A, STX3 and STX4. d | STX1A, EPIM/STX2 are synthesized as 34 kDa molecules, but only EPIM is released from the cell surface as a 30 kDa molecule. Mutation of His246 in EPIM/STX2 to Arg blocks secretion into the supernatant (Sup), whereas mutation of Arg246 to His in STX1A is sufficient to lead to extracellular secretion. TM, transmembrane. Figure parts c,d are modified, with permission, from REF. 46 © (2007) Company of Biologists.

The use of different motifs for distinct roles has been found in other proteins that function on both sides of the plasma membrane: PHI, which is a member of the sugar metabolism pathway, functions outside of cells as the morphogenic cytokine AMF and also possesses distinct structural domains for the distinct activities of PHI and AMF³⁶. Similarly, C-terminal truncation of thioredoxin (also known as adult T-cell leukaemia-derived factor (ADF)) enhances its mitogenic, cytokine-like activities, although its oxidoreductase function is compromised⁴⁴. However, such use of different motifs is not an absolute requirement for the successful integration of multiple distinct functions, as in the case of TTG, for which the consequences of a single enzymatic activity differ dramatically according to intra cellular or extracellular localization.

Unconventional secretion

Despite the diversity of functions and the apparent lack of significant primary sequence homology between the proteins that are listed above, these proteins lack exocytosis-targeting signal sequences but clearly exit the cell.

Examples of unconventional secretion

Although epimorphin/syntaxin 2 exists primarily on the cytoplasmic side of the plasma membrane⁴⁵, recent studies have defined some of the key protein motifs that are involved in unconventional secretion of epimorphin⁴⁶. When localized to the cytoplasmic surface, epimorphin/syntaxin 2 associates with synaptotagmin and annexin II, and when the cell is exposed to stress stimuli, the complex is translocated and released from the cell. Although much remains to be determined regarding the biogenesis of flipped epimorphin/syntaxin 2, these studies provide specific mechanistic information regarding how this process occurs. Similar mechanisms have been identified for other unconventionally secreted proteins^31,47,48.

Amphoterin/HMGB1 escapes the cell by at least two distinct mechanisms: passive release following necrotic cell death or active secretion in response to certain stimuli. When released from necrotic cells, HMGB1 functions as an immediate trigger for inflamma tion⁴⁹. Active secretion of HMGB1 can occur in response to inflammatory stimuli and is controlled by acetylation of the protein. Acetylation results in a shift in the intracellular equilibrium of the protein that favours the nucleus in unstimulated cells and the cytoplasm following activation^18,50. HMGB1 has been shown to accumulate into secretory lysosomes — Ca²⁺-regulated organelles that are released from the cell by exocytosis¹⁸.

A distinct mechanistic route for extra-cellular transport has been suggested for thioredoxin. Inside the cell, this enzyme catalyses thiol–disulphide exchange reactions, whereas it exerts cytokine-like and chemokine-like activities in the extracellular space⁵¹. Extracellular release of thioredoxin from T lymphocytes occurs rapidly following exposure to redox agents and is dependent on the redox-sensitive site of thioredoxin⁵¹.

How did unconventional secretion evolve?

Although not all of the proteins that are released by unconventional secretion have distinct but linked functions, unconventional mechanisms of secretion might be important in enabling dual but integrated functionality for single proteins. How might unconventional secretion be helpful, or even necessary, for this? In some cases, an alternative secretion pathway might be required when transport through the ER–Golgi results in protein modifications that interfere with the extracellular function of a protein, as has been shown for fibroblast growth factor 2 (FGF2)⁵². However, a need to avoid Golgi–ER processing cannot provide a universal rationale for invoking unconventional secretion. For example, forced transit of epimorphin/syntaxin 2 through the ER–Golgi results in glycosylated proteins that are nevertheless capable of directing morphogenesis⁴⁰. Perhaps a more general rationale might be that the lack of an exocytosis signal sequence and the use of unconventional secretory pathways by proteins that have paired functions allow for greater flexibility in compartmental distribution. Whereas the presence of a strong exocytosis signal in the protein would require extraordinary mechanisms by the cell to retain the protein, unconventional pathways might allow for flexible and dynamic regulation of protein localization in response to various internal and external stimuli, and thus might facilitate more intimate linkage of intracellular and extracellular functions. Unconventional secretion mechanisms might have developed early in evolution, before the formation of the ER–Golgi pathway. For these early cells, it would have been crucial to coordinate cellular function with intercellular communication. These mechanisms might have been retained because they allow for rapid connection between diverse processes.

Linking distinct functions

The above hypothesis predicts that there should be common mechanistic links between the intracellular and extracellular protein functions and the control of secretion, such as key post-translational modifications that code for retention in the cytoplasm compared with exportation, and environmental or cellular signals that trigger protein secretion. A number of mechanisms are involved in the transport of proteins across the plasma membrane by unconventional pathways (reviewed in REFS ^31,47,48), but defining the specific modifications that signal for these proteins to exit the cell has been delayed by the lack of clear consensus regions that govern the process. However, recent computational efforts have begun to address this problem (BOX 2). Identification of the specific protein modifications and secretion signals that trigger unconventional secretion is an important area for future investigation.

Box 2. Identifying unconventionally secreted proteins.

A simple motif that defines the proteins that are secreted by unconventional mechanisms has not been identified. However, several methods that use computational approaches have been developed to identify leaderless proteins, which might be secreted by unconventional pathways in eukaryotic⁵⁷ and prokaryotic⁵⁸ organisms. For example, Bendtsen et al. created a neural network that used sequence-derived features to identify such proteins; features include the presence of potential sites of post-translational modifications, predicted secondary structure, the abundance of charged residues, the presence of predicted propeptides and other transmembrane helices, and regions of low complexity. Using this network, they identified many proteins that are known to be secreted by unconventional mechanisms, including fibroblast growth factor (FGF)-family members, thioredoxin and galectin 1 (REFS ^57,58). An automated evaluation of known proteins or unknown protein sequences can be made at the SecretomeP 2.0 Server website.

A recent study has implicated caspase 1 as a mediator of unconventional protein secretion, and a screen to identify proteins that transit the plasma membrane in response to caspase 1 activation identified several proteins that are known to exit the cell by unconventional means. These proteins include annexin A2, macrophage migration inhibitory factor and high mobility group protein A2 (HMGA2) (REF. ⁵⁹).

Consideration of how relationships between intracellular and extracellular functions of single proteins might have developed can assist the identification of other members of this family. Studies of large, structurally homologous but mechanistically divergent protein families have suggested that new protein functions often evolve through an opportunistic process that is known as recruitment, in which the pre-existing structural features of an active site or ligand binding site are exploited for a new purpose^53,54. Evidence suggests that protein speciation often proceeds through inter mediates that have promiscuous functionality and are capable of binding multiple ligands and facilitating multiple biological processes⁵³. Seen in this way, the acquisition of dual topology would provide an additional mechanism to acquire multifunctionality.

For a molecule that originally evolved to function through the selective binding of a particular ligand from the array of potential intracellular binding partners, extracellular localization would result in exposure to a novel pool of potential ligands and allow the conscription of a pre-existing protein binding site for new functional interactions. In some cases, the intracellular and extracellular functions might be unrelated. In these cases, the bifunctional protein might be a transient evolutionary intermediate. Subsequent gene duplication and divergent evolution would lead to protein speciation and separate proteins would become specialized for individual functions⁵⁵.

By contrast, for proteins such as those described above, the topologically distinct functions of the proteins are linked by a common purpose, which provides a nat ural mechanism for integrated control of important cellular functions. In such cases, retention of both functions by a single protein species might have provided a selection advantage to the organism, with the result of preserving the bifunctionality of these proteins. This model predicts that, for proteins that maintain linked functions, the different domains that confer distinct functions would be preserved in evolution, as is seen for the epimorphin/syntaxin 2 secretion motif (FIG. 2c).

Conclusions

We have described examples of proteins that are joined by the common thread of dual topology and dual functionality to produce a unified tissue function. These proteins might function during epithelial morphogenesis to create the sidedness that provides functionality to polarized secretion, that links gene expression with chemotactic and inflammatory responses during wounding or that integrates cell survival with tissue structure and function throughout the maintenance of healthy tissues. The concept that single proteins could have distinct but linked functions on opposite sides of the plasma membrane is not widely recognized. However, it is important to remember that the idea of a single protein possessing distinct but linked actions in different intra cellular locations, as exemplified by β-catenin (a structural molecule at adhesion plaques and a trans cription factor in the nucleus), was once regarded as controversial. Through careful and detailed investigations of the inter dependence of function and localization, this concept has opened up new fields of study. Higher organisms have developed many mechanisms for increasing the complexity of a small genome, and the idea that a gene codes for a single protein that denotes a single function has been discarded following the recognition of the importance of gene splicing and post-translational modifications on protein function.

Many proteins coordinate distinct signalling pathways in the cells by having multiple functions, and the concept of ‘moonlighting’ was used to describe this phenomenon⁵⁶. Initially, this was an apt description, as the newly discovered function of an already defined protein seems to be secondary. However, this term gives the inevitable impression that one set of functions is more important than the others. We propose that this is not the case for the molecules described above. Although the locations of their distinct molecular activities are separated by the physical barrier of the membrane, their dual roles might coordinate a single tissue function and thus be equally important in an integrated overall purpose. We suggest that these proteins should instead be considered to be liaison proteins. This term might better reflect their shared function across the plasma membrane.

It is possible that many proteins might exist both inside and outside of the cell, showing distinct but related functions in these different cellular contexts. It would be intriguing to consider how many potentially serendipitous observations have been disregarded as a consequence of prior identification of a given protein in a different location and with a different function. The phenomenon of proteins with distinct functions inside and outside of the cell might not prove to be as common as alternative splicing or post-translational modifications, but given the importance of robust linkage of cellular functions and tissue processes, we expect that the linkage of function across the plasma membrane might be much more common and important than previously suspected.

Biographies

Derek Radisky carried out his Ph.D. research on yeast genetics at the University of Utah, Salt Lake City, USA, and did postdoctoral research in mammary morphogenesis and malignancy at Lawrence Berkeley National Laboratory, Berkeley, California, USA. He is currently investigating mechanisms of breast and lung malignancy at the Mayo Clinic, Jacksonville, Florida, USA.

Melody Stallings-Mann carried out her Ph.D. research at the University of Missouri, Columbia, USA, studying the role of retinol-binding proteins in early pregnancy. She worked as a postdoctoral fellow at Case Western Reserve University, Cleveland, Ohio, USA, and now works at the Mayo Clinic, Jacksonville, Florida, USA, where she investigates the role of signalling molecules and how inadvertent expression might lead to tumour initiation and growth.

Yohei Hirai carried out his Ph.D. research in biophysics at Kyoto University, Kyoto, Japan. His research has investigated substrate-protein interactions, the role of cadherins in cell adhesion and the function of epimorphin as a stromal morphogen. He is now at the Institute for Frontier Medical Sciences at Kyoto University, where he investigates the molecular mechanisms that underlie tissue morphogenesis.

Mina J. Bissell is a Distinguished Scientist at the Ernest Orlando Lawrence Berkeley National Laboratory, Berkeley, California, USA. She earned a B.A. in chemistry from Harvard College, Cambridge, Massachusetts, USA, and a Ph.D. in microbiology and molecular genetics from Harvard University. She was the Director of Cell and Molecular Biology and the Director of the Life Sciences until 2002 at Lawrence Berkeley National Laboratory. She is also on the affiliated faculty of the Comparative Biochemistry Group and three other graduate groups at the University of California, Berkeley, USA. Her laboratory investigates mechanisms that underlie tissue specificity, with particular emphasis on the role of the extracellular matrix and the microenvironment in mammary morphogenesis and breast cancer.