Lipocalin Receptors: Into the Spotlight

Evidence has been steadily accruing over time that a significant number of lipocalins interact with specific membrane receptors. The transfer of RBP:retinol across the cell membrane, faciliated by the elusive RBP receptor was for many years the archetypal example of a lipocalin:receptor mediated process. Megalin was subsequently identified as a low affinity/high capacity endocytic membrane receptor capable of binding a range of lipocalins including RBP.

Progress in the field has accelerated in recent years. A receptor for glycodelin has been found on T-cells and is thought to be involved in the downregulation of inflammatory processes. A receptor for lipocalin-1 has been cloned and shown to be a member of an undefined gene family of membrane proteins potentially possessing multiple transmembrane segments.

It is hoped that continuing advances in studying molecular interactions will allow the identification and cloning of other lipocalin receptors leading to a more complete understanding of how lipocalins participate in biological processes.

Introduction

Over the last three decades, there has been growing experimental evidence that an increasing number of lipocalins bind to specific cell surface receptors. To date and chronologically, the first reports in the literature included receptors for retinol-binding protein (RBP),¹ α-1-microglobulin (α-1M),² purpurin,³ β-lactoglobulin (Blg),⁴ epididymal retinoic acid binding protein (ERABP),⁵ insecticyanin,⁶ α-1-acid glycoprotein (AGP),⁷ odorant-binding protein (OBP),⁸ glycodelin,⁹ and lipocalin-1 (Lcn-1).¹⁰

The aim of this review is twofold; firstly to produce an up to date summary of work in the lipocalin receptor field; secondly to encourage innovative approaches to answering the many questions that remain on this topic. At this stage it is not known how many lipocalin receptors there are or whether they are related and how many families they represent. Thus the rest of the decade should be a period of excitement in the lipocalin receptor field.

The review will focus on the few systems for which there is a body of structural and functional data, which authenticate the presence of a receptor.

The RBP Receptor Story

RBP is perhaps the most widely understood of the lipocalins in terms of its structure and function (see Chapter 7). Its roles in the release of retinol from liver, and transport of this vitamin in blood are important for growth, differentiation, reproduction and normal vision. In healthy humans, RBP serum levels are typically in the region of 2 μM, with RBP:retinol complexed with another protein, transthyretin (TTR).¹¹ This 76 kDa complex, consisting usually of 4 molecules of TTR and 2 of RBP, is of sufficient size not be excreted in the glomeruli.¹² Once RBP releases its retinol, the resultant apo protein also has a much lower affinity for both receptor and TTR and is subsequently filtered in the kidneys and degraded in the urine. In the meantime, the retinol transferred into cells is bound to cellular retinol binding protein (CRBP), a 15 kDa protein, prior to metabolism and utilization.^13-15

Several schools of thought have developed on the exact mechanism of retinol release and entry into the cell. Some argue that retinol, being in equilibrium with RBP and extremely hydrophobic, readily partitions into the lipid phase of the membrane and thence to CRBP.^12,16-22 During studies on RBP gene-disrupted mice, a reduction in levels of serum retinol was observed and additionally in the first five months of life, animals exhibited visual impairment.²³ The same study showed that an enhanced vitamin A diet could restore vision and that the mice were viable, suggesting that other routes can exist for introducing vitamin A into the cellular metabolism. Later work by the same group demonstrated that the visual defects could be reversed by engineered expression of human RBP in the background of RBP-null mice,²⁴ indicating a role for RBP. The authors further suggest that at least in the visual system there appears to be the need for a membrane receptor.

Around the same time, two human siblings were reported with apparently undetectable levels of serum RBP which was put down to point mutations in their RBP gene.²⁵ Both suffered night blindness and mild retinal dystrophy but none of the other complications associated with clinical vitamin A deficiency.

Perhaps of some importance is the observation that mice obtain sufficient retinol during weaning to sustain them throughout their entire life cycle calling into question interpretations of data from mice models.²⁶ Additionally, there are indications that age and disease status may influence retinol metabolism. There is also evidence to suggest that very low intakes of retinol reduce retinol utilisation, in a manner consistent with maintaining adequate reserves for key processes (see ref. 27). Thus, one should bear in mind the different living conditions of native and laboratory animals, together with the evolutionary pressures which pertain to animals living in the wild.

For such reasons, a greater number of researchers were persuaded by the regulatory logic of an RBP:receptor mediated uptake system. This was borne out by indications of a specific receptor for RBP in the plasma membrane of many eukaryotic cells including the visual photoreceptor cells and placenta. The RBP receptor has been confirmed in Sertoli,²⁸ stellate,^29,30 peritubular pigment epithelial,³¹ embryonic carcinoma,³¹ choroid plexus³² and a variety of tissue culture cells.^33,34 The placental receptor showed high affinity for the holo-form of RBP but not for the apo-form.³⁵ Purification studies suggested it had a molecular weight in the region of 55kDa.³⁶ More recent work with RBP- knockout mice has supported a natural receptor-mediated retinol uptake system, which does not involve endocytosis or free diffusion of retinol.^23,24,37 Other lipocalins were unable to bind to this receptor indicating specificity. Since then it has been found that there are also specific membrane receptors for other lipocalins.^4,38,39

The subsequent fate of RBP upon binding to the cell surface has been a source of some debate. Some reports show that the retinol is released and the apoprotein remains in the extracellular compartment.^40-42 Other reports say that the RBP:retinol complex is internalized.^43-46 Evidence has been presented to suggest that cells which endocytose the RBP:retinol complex do so to create vitamin A stores that can be rereleased into the blood stream when required.^47,48 For that reason, that may be the same explanation for evidence that RBP can be synthesised in tissues other than the liver.⁴⁹ Another possibility is that the difference is due to the various biological systems and methods employed. Heller¹ used intact, isolated pigment epithelium cells recovered from bovine eyes and the Findlay group⁴² used placental vesicles. Groups characterising the RBP:receptor interaction as an endocytic process were all using cultured cells. It is possible that both routes occur, defined by tissue type.

During these two decades, the research focus has shifted towards a more detailed understanding of the RBP:receptor interaction and the process has reinforced the evidence for a receptor.

Using site-directed mutagenesis, Sivaprasadarao and Findlay⁵⁰ delineated the roles of the loop regions present around the mouth of the RBP beta barrel in recognising both the RBP membrane receptor and TTR. Their results demonstrated that RBP interacts with both TTR and the receptor via loops “CD” and “EF.” These binding sites, however, were overlapping rather than identical and there was the possibility also of an additional contact with TTR via loop “AB.” One of the implications of these results is that RBP, when bound to TTR, cannot bind simultaneously to the receptor. Confirming these observations, Melhus and colleagues⁵¹ subsequently used monoclonal antibodies to try and block both the TTR and RBP receptor binding sites. Epitope mapping using synthetic peptides corresponding to RBP loop regions reported the TTR and RBP binding sites in agreement with the earlier study.⁵⁰ Structural data, produced afterwards, confirmed the nature of the RBP:TTR interaction as derived from the protein engineering studies.^52,53 The interaction of RBP and TTR from a structural perspective is discussed in more detail in Chapter 7.

Further studies focussed on the “CD” loop of RBP (comprising amino acids G59-D68 of the sequence), which is crucial for receptor recognition and interaction. Sundaram et al⁵⁰ demonstrated that this loop and the ability to recognise the RBP receptor, could be transferred successfully to the equivalent position in ERABP⁵⁴ and in a quite different lipocalin such as MUP (Redondo, Vouropoulou and Findlay, in preparation).

They further extended the notion of Sivaprasadarao and Findlay that the RBP receptor is essential for transfer of retinol from extracellular RBP to cellular retinol-binding protein (CRBP) inside the cell (See fig. 1). Membranes lacking the receptor (e.g., erythrocytes) and heat-denatured systems are ineffective. Whether this occurs via the receptor itself or a separate protein system is recruited for the transfer, remains to be determined. CRBP is a member of the fatty acid-binding protein family (FABP) which is structurally related to the lipocalins. Intracellular levels of retinoids are regulated by CRBP which has been suggested to exert in a buffer-like role mediating further metabolism and utilization.

Figure 1

Receptor-mediated uptake of retinol from retinol binding protein. Schematic representation of the events proposed to facilitate transfer of retinol across the cell membrane. Retinol binding protein (shown at the top of the bilayer) complexes with retinol (more...)

Recent observations⁵⁵ suggest that CRBP itself also exhibits specific membrane-association characteristics indicating the existence of a protein receptor, which may or may not be related to retinol metabolising enzymes, supporting the early results obtained by Sundaram et al.⁵⁶

It is worth pointing out that this is a unique delivery/uptake system not yet seen elsewhere in eukaryotic biology but the principle does exist in prokaryotes, although the extracellular and intracellular binding proteins and the membrane receptor/transporter are quite different.⁵⁷

Megalin

Megalin, a member of the low density lipoprotein receptor family, was the first, but nonspecific, lipocalin receptor identified. It is a 600-kDa type-1 cell surface protein, classically consisting of a single transmembrane domain, a large amino-terminal extracellular domain and a short carboxy-terminal cytoplasmatic tail.⁵⁸ The detailed structure, expression, and physiological properties of megalin have been studied in great detail over the past few years and there are numerous recent reviews (refs. 59-65). Megalin mediates endocytosis of a large variety of ligands including vitamin-binding proteins and other carrier proteins, lipoproteins, hormones and hormone receptors, drugs and toxins, enzymes and enzyme inhibitors, immune- and stress-response-related proteins and some other proteins, eg. cytochrome C (for a review see ref. 58). Therefore, it is not unexpected to find megalin involved in the cellular uptake of lipocalins. First evidence for a role of megalin in cellular uptake of RBP came from megalin deficient mice. These mice showed a lack of RBP production in their renal proximal tubules and instead revealed highly increased urinary excretion of RBP and retinol, indicating that glomerular filtered RBP-retinol complexes escape uptake by proximal tubules.⁶⁶ A direct interaction of megalin and RBP was confirmed by surface plasmon resonance analysis and partial inhibition of RBP uptake by a polyclonal megalin antibody in Brown Norway rat yolk sac epithelial cells.⁶⁶

Further investigations on the megalin knock-out mice revealed that megalin binds and mediates uptake of a number of other lipocalins present in mouse plasma and urine, including alpha-1 microglobulin, major urinary protein 6 and odorant-binding protein IA.⁶⁷ Because in the mouse system, many of the proteins that were found to bind megalin were plasma carriers for lipophilic compounds (e.g., fatty acids, odors, vitamin A and D metabolites), it was suggested that megalin might be essential to prevent urinary loss of lipids bound to small carrier proteins. This more general function is further supported by the fact, that the affinities of megalin for lipocalins and other binding proteins is only 0.1 to 1.8 μM, indicating a low affinity but high capacity receptor.⁶⁸

LIMR

In a search for proteins that interact with the lipocalin Lcn-1 using phage-display technology, the Redl group identified a novel receptor , which was termed LIMR (Lipocalin-1 Interacting Membrane Receptor;¹⁰). Lcn-1 is a lipocalin with broad ligand binding specificity,⁶⁹ which structurally differs from other lipocalin members by its extremely wide ligand cavity framed by a set of four loops at the open end. This cavity extends deeply into the barrel structure and ends with two distinct lobes.⁷⁰ Lcn-1 is thought to act as a physiological scavenger of potentially harmful hydrophobic molecules and was recently found to be another member of the lipocalin subgroup of siderocalins, since it binds bacterial and fungal siderophores.^71,72

LIMR is essential for the internalization of Lcn-1 in human NT2 cells,⁷³ and thus functions as a endocytic receptor similar to megalin. However, the structural composition of LIMR appears to be completely different from that of megalin and other endocytic receptors. LIMR is a 55 kDa protein, which consists of a short extracellular domain, nine putative transmembrane domains interrupted by a large intracellular loop and an intermediate length cytoplasmatic tail.^73,74 Phage-display experiments and interaction analyses of purified recombinant peptides in solution revealed that LIMR binds Lcn-1 via the N-terminal region.¹⁰ These results are supported by the fact that a polyvalent antiserum raised against the LIMR N-terminus abrogates uptake of FITC-Lcn-1 in NT2 cells (Redl, unpublished).

LIMR appears to constitute a novel family of endocytic receptors, whose members can be found in a wide variety of organisms including Mus musculus, Fugu rubripes, Caenorhabditis elegans, Drosophila melanogaster, Anopheles gambiae, Dictyostelium discoideum and Arabidopsis thaliana. Humans and mice, each have two closely related proteins in addition to up to 7 other putative homologues. In humans there is a LIMR orthologous protein called Dif14, which is encoded on chromosome 7q36, whereas LIMR is encoded on chromosome 12p11. In mouse there are also two closely related proteins, one is highly similar to human LIMR, whereas the other, Lmbr1, is more similar to human Dif14. It is interesting to note, that sequence comparison of human and mouse LIMR-related proteins reveals a relatively lower degree of conservation in the N-terminal region of the proteins. For example, whereas the overall sequence conservation of human LIMR and Dif14 is 58.3%, amino acid identity in the N-terminal region of the respective proteins is only 12.5%. Assuming that the N-terminal parts of these proteins are responsible for receptor-ligand interaction, as shown with LIMR, these results might indicate that the orthologous proteins found within one species interact with different target proteins.

A very recent study demonstrated that the LIMR gene is one of the genes that are highly induced in response to dietary iron-deprivation in rat duodenum.⁷⁵ An enhanced cellular uptake of siderocalins might indeed play a role in iron homeostasis in the gut since they are known to participate in the iron-depletion strategy of the immune system and are able to mediate iron absorption by some cell types^75,76). Thus, it is possible, that LIMR or its orthologues have a function in the internalization of siderocalins, in general. However, the exact role of these receptors in binding of additional lipocalins or other transport proteins remains to be determined.

Glycodelin

Glycodelin is implicated in a variety of processes within the reproductive axis (reported in detail in Chapter 11). Specifically, in the case of females, at least one isoform of glycodelin is capable of binding to sperm in order to inhibit egg-binding.⁷⁷ More generally, it is implicated in regulation of inflammation, specifically by downregulation of T cells. This is achieved by the binding of glycodelin to CD45 (a major protein tyrosine phosphatase receptor) and other glycosylated entities on the surface of T-cells in a lectin-like manner.⁷⁸ The glycosylation state of glycodelin is critical to the varying roles of the three isoforms found.^79,80 Researchers have proposed that glycodelin alters the local balance between tyrosine kinases and phosphatases thereby attenuating T cell receptor signalling. The net effect is to damp down the immune response. The exact role of CD45 in T cell activation is unclear at this stage. Interestingly, glycodelin has been observed in the cumulus/corona cells which accompany the ovum during ovulation (which themselves do not contain mRNA for glycodelin⁸¹) implying the presence of an uptake pathway.⁸¹ Which receptor is potentially involved remains to be determined.

Lipocalin-2

The functional characterisation of lipocalin-2 (Lcn2) has advanced significantly in recent years. Proposed roles include that of an antibacterial agent⁷⁶ as well as involvement in cellular differentiation.⁸² Additionally, both transcriptomic and proteomic methodologies have detected significant increases in the levels of Lcn2 related to various cancers.^83,84

As is the case with many lipocalins, Lcn2 is taken up by the megalin scavenging receptor.⁸⁵ Interestingly, the tightness of interaction is much greater than for other lipocalins so far examined, with Lcn2 displaying a dissociation constant that is approximately three orders of magnitude lower when compared to lipocalins such as RBP and MUP. It has been suggested that this might be explained in part by the net positive charge displayed by Lcn2.⁸⁵

However, interaction with megalin is not sufficient to explain some of the functional behaviours of Lcn2, especially given that Lcn2 is present in a broader range of tissues than the endocytic receptor. A recently published paper documented the involvement of Lcn2 in regulation of cell phenotype by acting as an inhibitor of the intracellular pathway responsible for E-cadherin degradation.⁸² The effect is enhanced by the presence of siderophore:iron complexes but not when the iron is substituted by gallium.⁸² Furthermore, supplementation of cell systems with free iron does not facilitate this effect and at higher levels increased degradation of E-cadherin is observed.⁸² Given that megalin does not distinguish between apo and holo forms of Lcn2, nor is it specific as to which proteins it endocytoses, one would anticipate another mode of modulating this intracellular pathway. Further work is required to delineate the precise role of this lipocalin and whether a membrane receptor other than megalin is implicated in mediating its observed effects.

The Future of Lipocalins and Their Receptors

Perhaps the most consistent and difficult challenge facing researchers working in the field of lipocalin receptors is the very nature of the receptors themselves. Membrane proteins, whilst accounting for as much as 30% of the genome, are more difficult to detect, purify and characterize than soluble proteins. This has delayed the molecular biology substantially. It is therefore not surprising that lipocalin receptors have proved so elusive in revealing their identities.

As greater understanding of the evolution of the lipocalins is developed (See Chapter 2), it will be interesting to observe the broader evolution of functional systems.

The eye is an excellent example of this. The more complex eyes found in chordates rely on a sophisticated system, requiring the delivery of retinol by RBP to the RPE by a receptor-mediated process. The chromophore can only be regenerated in the RPE. It would be reasonable to infer that rhodopsin, RBP, iRBP, CRBP and the RBP receptor have all evolved within a similar time frame to help facilitate chordate vision in a way that offers clear regulatory advantages. The same is likely to be even truer of the reproductive and foetal systems.

Some researchers have speculated as to the existence of a structurally related family of membrane receptors, specific for the lipocalins.⁶⁸ This maybe likely for lipocalins that are similar in terms of sequence and function. However, the scope of lipocalin function is so diverse that one cannot rule out more than one class of specific lipocalin receptors. Moreover, it seems likely that the roles of these receptors will vary to include signal transduction, endocytosis, and transport. The presence of additional subunits would not be unexpected, nor would be the presence of a substantial quaternary structure. The one definitive receptor class now identified, raises a number of interesting fundamental issues because its putative structure is so atypical of an endocytic receptor.

Artificial evolution of the lipocalin fold gives further indication of the potential for lipocalin:receptor interaction. An engineered lipocalin was recently produced with novel specificity for CTLA-4, a receptor on the surface of T cells which in principle could allow lipocalins to behave has receptor agonists/antagonists.⁸⁶

As our knowledge of lipocalins and their receptors continues to evolve, we anticipate a greater understanding of the functional significance of certain lipocalins.

Acknowledgements

The authors wish to acknowledge and thank Prof. Markku Seppälä for helpful discussions regarding glycodelin.

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