The LDL receptor‐related protein (LRP) family: An old family of proteins with new physiological functions

Abstract

The low‐density lipoprotein (LDL) receptor is the founding member of a family of seven structurally closely related transmembrane proteins (LRP1, LRP1b, megalin/LRP2, LDL receptor, very low‐density lipoprotein receptor, MEGF7/LRP4, LRP8/apolipoprotein E receptor2). These proteins participate in a wide range of physiological processes, including the regulation of lipid metabolism, protection against atherosclerosis, neurodevelopment, and transport of nutrients and vitamins. While currently available data suggest that the role of the LDL receptor is limited to the regulation of cholesterol homeostasis by receptor‐mediated endocytosis of lipoprotein particles, there is growing experimental evidence that the other members of the gene family have additional physiological functions as signal transducers. In this review, we focus on the latest discovered functions of two major members of this family, LRP1 and megalin/LRP2, and on the newly elucidated physiological role of a third member of the family, MEGF7/LRP4, which can also function as a modulator of diverse signaling pathways during development.

• LRP1
• megalin/LRP2
• MEGF7/LRP4

Introduction

There are seven members of the low‐density lipoprotein (LDL) receptor superfamily that are structurally closely related: LRP1, LRP1b, megalin/LRP2, LDL receptor, very low‐density lipoprotein receptor (VLDL receptor), MEGF7/LRP4, and LRP8/apolipoprotein E (apoE) receptor 2.

The founding member of the family, the LDL receptor, regulates cholesterol homeostasis by receptor‐mediated endocytosis of cholesterol‐rich LDL particles, and mutations of the LDL receptor gene were shown to underlie the lipid disorder familial hypercholesterolemia. In contrast to initial assumptions, the LDL receptor seems to be the only member of the family with a role limited to the uptake of lipoproteins (for review see 1,2) as recent studies showed that the other members fulfill important functions outside lipid metabolism.

The second member of the family cloned was the low‐density lipoprotein (LDL) receptor related protein (LRP1) 3. It is a large endocytic receptor widely expressed in several tissues and known to function in areas as diverse as lipoprotein transport, regulation of cell surface protease activity, control of cellular entry of bacterial toxins and viruses, and protection against atherosclerosis by modulation of platelet‐derived growth factor receptor‐β (PDGFβ) signaling in the vascular wall 4–7.

Megalin/LRP2, another multifunctional receptor, is the largest member of the family with a size of approximately 600 kDa. It was first described as a scavenger receptor due to its multiligand binding properties. It has been shown to be the antigenic determinant for Heymann nephritis in rats and is important for the reabsorption of various molecules in the proximal renal tubule. However, it was also demonstrated that megalin, like LRP1, has additional functions in cellular signaling transduction 8–10.

Beside LRP1 and megalin/LRP2, two other members of the LDL receptor family, apoER2 and VLDL receptor, have been recognized as signal transducers. In particular, they control signaling pathways that are critical for brain development, which have been reviewed recently 11,12.

This review focuses on novel knowledge about the functional aspects of LRP1 and megalin/LRP2 in endocytosis and signal transduction. In addition, we will discuss recent work which establishes another member of the LDL receptor superfamily, MEGF7/LRP4, as an important modulator of diverse signaling pathways during development. The function of the remaining member, LRP1b, is still largely unknown to date and will not be a subject of this review.

Key messages

• LRP1 is an endocytic receptor and has essential signaling functions.

• Megalin/LRP2 is an endocytic receptor that regulates the availability of extracellular signaling molecules.

• MEGF7/LRP4 controls limb development.

LRP1

LRP1 (also known as CD91, or α2macroglobulin receptor, α2MR) is a ubiquitously expressed type 1 transmembrane receptor 3. The active receptor protein is derived from a 600‐kDa precursor processed by furin and consists of an 85‐kDa membrane‐bound carboxyl fragment (intracellular and transmembrane domains) and a noncovalently attached 515‐kDa amino‐terminal fragment (extracellular domain) 3,8 (Figure 1). LRP1 is one of the most multifunctional members of the family 13. It has so far been implicated in two major physiological processes: endocytosis and regulation of signaling pathways. Through its extracellular domain, LRP1 interacts with and mediates endocytosis of more then 40 different ligands ranging from lipoproteins, extracellular matrix proteins, protease/protease inhibitor complexes and viruses, to cytokines and growth factors 14. This wide range of ligands recognized by LRP suggests that it plays diverse biological roles including lipid metabolism, cell growth, migration, and tissue invasion. The remarkable degree of cross‐species identity conserved in the LRP amino acid sequence, and the failure of blastocysts to transform into embryos after targeted disruption of the LRP gene in the mouse 15 highlight the biological importance of this receptor.

Figure 1. Structure of MEGF7, LRP1 and megalin.

In addition to its role as an endocytic receptor 13,16, recent work also demonstrated that LRP has essential signaling functions. These functions have been described in the vessel wall, in neurons, and in the lung amongst other tissues. In the vessel wall, LRP1 has a major role in controlling vascular smooth muscle cell (VSMC) proliferation and protects from atherosclerosis. Mice that lack LRP1 in vascular smooth muscle cells demonstrated hyperplasia of the aortic wall, disruption of the elastic layer, and increased susceptibility to develop atherosclerotic lesions 5. In addition to hyperplasia of the aortic media, these lesions are characterized by extensive and rapid foam cell formation that leads to the complete occlusion of the aorta and to the death of the animal. The protective effect of LRP1 seems to be exerted mainly by controlling certain signaling pathways, one of which has been identified: hyperplasia of the aorta was accompanied by hyperactive PDGF signaling with increased steady‐state levels of PDGF receptor β and augmented phosphorylation of extracellular regulated kinase ½ (Erk1/2) 5–7.

Activation of PDGF signaling has been described to occur secondary to transforming growth factor β (TGFβ) activation 17–19. Furthermore, LRP1 was recently reported to be identical to TGFβ receptor (V), which is coexpressed together with TGFβ receptor I, II, and III 20. Thus, additional interactions between LRP and growth factor receptors in the vascular wall may tightly regulate signaling processes that protect against atherosclerosis.

In addition to the contribution of LRP to protect against atherosclerosis, recent work revealed a novel role for LRP and its ligand the tissue plasminogen activator (tPA) in modulating vascular tone 21 and in regulating the blood‐brain barrier (BBB) permeability 22. tPA regulates vascular contractility through the low‐density lipoprotein receptor‐related receptor (LRP1), and this effect is inhibited by plasminogen activator inhibitor type 1 (PAI‐1). The authors described that tPA‐mediated vasocontraction involves the coordinated interaction of LRP with the integrin alpha(v)beta3,23. However, the mechanism underlying these interactions and the nature of the signals transduced is unknown. Yepes et al. 22 reported that tPA regulates BBB permeability via a process dependent upon LRP. The regulation of cerebrovascular permeability is critical for normal brain homeostasis and protects the central nervous system (CNS) from the entrance of potentially toxic substances present in the blood. The authors demonstrated that tPA directly induces opening of the BBB, and this is blocked by antibodies to LRP1 and by the lipoprotein receptor antagonist, the receptor‐associated protein (RAP), suggesting a LRP‐mediated process 22.

LRP1 has also been shown to have a major pathophysiological role in the central nervous system (CNS). It is highly expressed in neurons 24,25 where it interacts with several neuronal proteins such as postsynaptic density protein 95 (PSD‐95) and N‐methyl‐D‐aspartate (NMDA) receptor subunits 26. Glutamate is the main excitatory transmitter in the CNS and is known to play an important role in neuronal degeneration 27,28. Furthermore, in in vitro models, LRP regulates calcium signaling, an important second messenger for neuronal response to excitatory neurotransmitters such as glutamate 29. Activated α2‐macroglobulin (α2M), a ligand of LRP, reduced through an LRP‐mediated pathway the calcium responses to NMDA and NMDA receptor expression 30. The generation of genetically modified mice that lack LRP in neurons has proved the in vivo importance of LRP functions in this cell type. Animals display severe behavioral and motor abnormalities with tremor, ataxia, hyperactivity and finally cachexia and premature death 26.

In the lung, a new role for LRP1 as a regulator of the inflammatory response has been suggested recently 31. The authors reported a model in which the surfactant proteins A and D (SP‐A and SP‐D), by binding to the signal inhibitory regulatory protein α (SIRPα), activate the tyrosine phosphatase SHP‐1 resulting in downstream blockade of signaling through Rous sarcoma virus (src)‐family kinases and P38 map kinase and a general suppression of inflammation. On the other hand, when SP‐A and SP‐D interact with foreign organisms, apoptotic cells, or cell debris, presentation of these proteins in an aggregated state to cell surface calreticulin and LRP1 on alveolar macrophages initiates phagocytosis, along with an inflammatory response. LRP1‐dependent phagocytosis does not take place only in pulmonary macrophages but also in phagocytic cells of other tissues 32. For instance, the system calreticulin/LRP is also involved in apoptotic cell removal, a critical process for development, tissue homeostasis, and resolution of inflammation 33.

Like a wide variety of receptors and other plasma membrane proteins, LRP1 has been found to shed its extracellular domain and a soluble form of LRP can be detected at nM concentrations in human plasma 34,35. The shed LRP contains the α‐chain (515 kDa subunit) and a 55 kDa fragment of the β chain (85 kDa subunit), revealing that proteolysis occurs in a membrane‐proximal region 34. The enzyme responsible for the extracellular cleavage seems to be cell‐dependent and was found to be the protease BACE1 in neurons 36 and a metalloproteinase in hepatocytes 34. The soluble form is present in the plasma of mammals, and also of birds, reptiles, and molluscs 37. In most cases, the physiological significance of receptor ectodomain shedding remains undefined. In the case of LRP1, the soluble form maintains the ligand binding characteristics of cellular LRP1 and can act as a competitive inhibitor of ligand uptake by cell‐surface‐bound LRP1.

The underlying signal transduction mechanisms of how LRP modulates or participates in signaling pathways are not fully understood. One mechanism, most likely, involves cleavage of the receptor within the plane of the membrane segment. The released intracellular domain (LRP‐ICD) of about 12 kDa can translocate to a new location within the cell where it may elicit the physiological response 38. This mechanism is called regulated intramembrane proteolysis (RIP). RIP is a process whereby a transmembrane protein can be cleaved within the plane of the membrane to liberate a cytosolic fragment that in some instances enters the nucleus and regulates transcription of target genes 39. Several proteins, including the amyloid precursor protein (APP), Notch, a plasma membrane receptor that controls cell fate decision throughout development, the receptor tyrosine kinase ErbB‐4 40, the hyaluronan receptor CD44 41, sterol regulatory element binding proteins (SREBPs) 42, activation transcription factor 6 (ATF6) 43, inositol requiring 1(Ire1) 44, and cadherin 45, have been shown to undergo this proteolytic processing. In most cases, the intramembranous cleavage is exerted by the presenilin (PS)/γ‐secretase complex. In the case of LRP, the receptor's intracellular domain appears to be also released from the plasma membrane by a proteolytic cleavage involving the presenilin‐containing γ‐secretase complex 38. However, neither the cleavage sites nor the potential target genes for the 12‐kDa fragment released have been identified, and the function of the LRP‐ICD once released from the parent molecule is unknown. The LRP‐ICD contains many potential signaling motifs: two NPxY motifs, the distal one overlapping with an YXXL motif, and two dileucine motifs. The YXXL might be the most dominant endocytosis signal 46. The two NPxY motifs are capable of interacting with cytoplasmic adaptor proteins and scaffold proteins, such as disabled protein 1(Dab1), FE65, JIP1, PSD‐95, Shc and CED‐6/GULP 6,7,47–51. Recently, in vitro studies 52 demonstrated that the LRP‐ICD appears to translocate to the nucleus where it colocalizes with the histone actetyltransferase Tip60, a transcription modulator that has numerous functions, including a role in linking the proteolytic cleavage of APP to transcriptional activation 53,54. This observation suggests that the LRP‐ICD may impact transcriptional activity of the APP‐Tip60 complex and sheds new light on the function of LRP as a transcriptional modulator. In order to molecularly dissect the function of the different motifs in endogenous LRP in vivo, Roebroek et al. 55 introduced mutations into the furin proteolytic cleavage site and into the NPxY motifs in the LRP‐ICD. Mutation of LRP in the proximal NPxY motif of the cytoplasmic tail and in the furin cleavage site caused liver phenotypes with perinatal death of the animal due to destruction of the liver and a selective enlargement of Kupffer cell lysosomes without effect on animal survival, respectively. Surprisingly, mutation of the distal NPxY motif within the tail of LRP did not cause any obvious phenotype 55.

In conclusion, LRP is a large, multifunctional, endocytic receptor that is a member of the LDL receptor superfamily. This complex receptor is widely expressed in most cell types and is especially abundant in vascular smooth muscle cells (SMCs), hepatocytes, and neurons. Not only does LRP1 effectively regulate the levels of several molecules by binding and facilitating their delivery to lysosomes for subsequent degradation, but clearly it participates in signaling functions essential for cell migration, cell proliferation, and regulation of vascular permeability. LRP1 may participate in these activities as a coreceptor and/or by interacting with a number of adaptor proteins involved in signaling pathways via its intracellular domain. The identification of in vivo events modulated by LRP1 requires further investigation. However, these events may serve as new targets for the treatment of different cardiovascular and neuronal disorders.

LRP2/megalin

Megalin (also known as LDL receptor‐related protein 2, LRP2, and glycoprotein 330, gp330) was originally identified as the autoantigen in an animal model of autoimmune kidney disease, namely Heymann nephritis, a membranous glomerulonephritis 56. Cloning and sequencing of its gene revealed the close similarity of the transmembrane receptor with members of the LDL receptor family of lipoprotein receptors 57, into which megalin was then included (Figure 1). Functional analysis showed that megalin is an endocytic receptor that binds and internalizes a variety of ligands including protease‐protease inhibitor complexes, vitamin‐vitamin binding protein complexes, other hormones, and lipoproteins (Table I). It is expressed at the apical surface of epithelial borders and is also found intracellularly in endosomes 58, the main sites of expression being the yolk sac and anterior neuroepithelium in embryos and the proximal renal tubule and intestinal epithelium in the adult organism 59. As recent studies showed, the endocytic uptake mediated by megalin does not simply serve cargo transport into the cell but is part of superordinate regulatory processes, e.g. of systemic vitamin homeostasis, and controls the availability of hormones and related first messengers in certain organ systems 60.

Table I. Extracellular ligands of megalin.

Ligand	Reference
Vitamin D/vitamin D binding protein complexes	61
Vitamin A/retinol binding protein complexes	63
Vitamin B12/transcobalamin complexes	64
Thyroxine/transthyretin	80
Sex steroids/sex hormone binding globulin complexes	66
BMP4	10
Low‐density lipoproteins	81
β very low‐density lipoproteins	82
Lp(a)	83
Apolipoprotein H	84
Apolipoprotein J	85

A mouse model with targeted disruption of the megalin gene first demonstrated both the receptor's essential role in embryonic development and in vitamin homeostasis 8,61. Conventional targeted deletion of megalin leads to holoprosencephaly, a midline defect of the forebrain and other cephalic structures, in homozygously affected mice 8. Initially it was discussed that deficient transport of cholesterol containing lipoproteins from the maternal circulation to the embryo could be causative to the defect. This hypothesis was supported by the fact that cholesterol deficiency from other reasons also leads to holoprosencephaly in the developing mouse embryo 62. Recent studies, however, elucidated a different mechanism underlying the malformation 10. It was found that in an animal model where megalin is still present in the yolk sac but absent in the embryo proper, the same neural phenotype occurs like in the conventional knockout‐model, ruling out deficient lipoprotein transfer from the maternal circulation as a cause. In addition it could be shown that megalin binds and internalizes the signaling factor BMP4, which is subsequently degraded in the lysosome. Loss of megalin leads to the accumulation of BMP4, which is a negative regulator of another extracellular signaling molecule, namely sonic hedgehog, Shh. Thus, Shh activity is reduced in the developing forebrain in megalin‐deficient animals, a defect that—like enhanced BMP4 signaling—has been shown to cause holoprosencephaly itself 62. So in conclusion, the study demonstrated that endocytic processes mediated by megalin can serve to limit the availability of a signaling factor in a tissue where it has crucial functions in regulating development. The megalin‐deficient animal model helped to elucidate another important systemic function of the receptor. Loss of megalin in the proximal renal tubule leads to greatly enhanced excretion of 25‐hydroxy‐vitamin D and vitamin D binding protein (amongst others) with the urine 61. Vitamin D deficiency and corresponding bone mineralization defects are the consequences of the reduced vitamin reuptake by megalin.

The endocytosis of vitamin D/vitamin D binding protein complexes is necessary to maintain adequate systemic vitamin D levels and to enable vitamin D activation by 1‐hydroxylation. In a similar way, vitamin A and vitamin B12 in complexes with their respective binding proteins are reabsorbed by megalin from the primary urine 63,64. Tissue‐specific targeted recombination helped to show that it is indeed loss of megalin function in the proximal tubule that leads to the disturbance of systemic vitamin metabolism, demonstrating a role for megalin in maintaining adequate vitamin levels, in addition to its previously mentioned role in limiting the availability of other signaling factors by endocytosis and delivery to lysosomal degradation 65.

Another direct link between endocytosis and signaling functions of megalin was described for the effect of sex steroids in the developing reproductive tracts of mice. Analysis of megalin‐deficient animals revealed that in addition to the previously described defects homozygous loss of megalin leads to malformations both in the female and in the male reproductive organs 66. Defective endocytic uptake of sex hormone/sex hormone binding globulin complexes by megalin, and resulting loss of sex hormone signaling in target cells, was identified as the underlying molecular defect.

This discovery is of particular interest because for the first time it was shown that a specific effect of steroid hormones was not induced by diffusion of the free hormone through the cell membrane but that endocytic uptake of the hormone and its binding protein by a transmembrane receptor was necessary. In addition to this remarkable new insight into steroid metabolism, it also demonstrated a direct role for megalin in signaling processes on the level of the receiving cell.

Another direct cellular signaling mechanism has been proposed for megalin in the epithelium of the proximal renal tubule, where the megalin extracellular domain can be shed in a metalloprotease‐dependent manner 67. The resulting membrane‐bound C‐terminal fragment accumulates when the γ‐secretase complex, which mediates the intramembranous cleavage of several transmembrane proteins, is inhibited 67. This suggests that the intracellular domain of megalin can be released from the plasma membrane by regulated proteolysis in a Notch‐like manner as it has been demonstrated for LRP1 38. The subsequent fate of the megalin intracellular domain has not been examined so far, but its interaction with intracellular adaptor proteins would allow it to regulate the assembly or subcellular localization of signaling complexes. Of particular interest in this context is the interaction of megalin with the newly identified adaptor protein megalin‐binding protein, which on its part binds Skip, a component of the vitamin D transcriptional complex 68. Whether the megalin ICD has a direct role in transcriptional regulation or how it might influence other intracellular signaling pathways remains to be elucidated.

In summary of our present knowledge, megalin is an endocytic receptor that regulates the availability of several extracellular signaling molecules in different ways. It can limit their activity by endocytic uptake and delivery for lysosomal degradation as shown for BMP4; it can regulate systemic levels by reuptake at exterior boundaries as shown for vitamin D and other vitamins in the epithelium of the proximal renal tubule; and it can deliver signaling molecules to target cells by endocytosis of the active messenger as shown for sex hormones in the reproductive tracts of mice. These different molecular functions of megalin define it as a lipoprotein receptor at the intersection of endocytosis and signaling.

LRP4/MEGF7

MEGF7 (multiple EGF‐like domain, LDL receptor‐related protein 4, LRP4) was identified during a motif trap screening 69 of gene encoding for proteins with multiple epidermal growth factor (EGF)‐like motifs in human brain cDNA libraries.

It possesses a ligand binding type repeat, EGF‐precursor homology domains, an O‐glycosylation domain, a transmembrane domain, and a cytoplasmic tail (Figure 1).

MEGF7/LRP4 appeared to belong to the LDL receptor family because the domain organization and the amino acid sequence of MEGF7 showed significant similarities to those of members of the LDL receptor family. Similarly to LRP1, megalin and all other members of the family, it possesses an NPxY sequence in its cytoplasmic domain, a tetraamino acid motif that has been shown to mediate the coupling of these receptors to both the endocytic machinery 70 and to a wide range of intracellular signaling cascades (Figure 1) 71. The structural organization of the extracellular domain of MEGF7/LRP4 resembles that of Lrp5 and Lrp6, two more distantly related members of the LDL receptor superfamily that are likely to function as coreceptors for the Wnt proteins 72,73.

MEGF7/LRP4 is expressed, amongst other locations, in migratory primordial germ cells (PGCs) in the hindgut and the dorsal mesentery of E9,5 embryos, and in germ cells in the genital ridges of male and female E10,5–13,5 embryos. MEGF7/LRP4 is also expressed in spermatogonia of the neonatal and adult testes and in the immature oocytes and follicular cells of the adult ovary. The absence of MEGF7/LRP4 expression in blastocysts, embryonic stem cells, and embryonic germ cells suggests that MEGF7/LRP4 is a molecular marker that distinguishes germ cells from embryo‐derived pluripotent stem cells 74.

The biological function of MEGF7/LRP4 has so far remained obscure. To gain a first insight of how MEGF7/LRP4 may function, Johnson et al. 75 have knocked out the gene in mice using disruption of MEGF7/LRP4 by homologous recombination in embryonic stem cells. Homozygously MEGF7/LRP4‐deficient mice present with growth retardation and polysyndactyly, a phenotype that is characterized by the fusion and duplication of digits at both the fore and hind limbs. The authors report that in MEGF7/LRP4‐deficient mice, the phenotype is combined with a mild and only partially penetrant form of craniofacial and tooth development abnormalities. The phenotype manifests itself at the early stages of embryonic limb bud development (E9,5), when the apical ectodermal ridge (AER), the center that regulates limb outgrowth at the extremity of the limb and the principal site of MEGF7/LRP4 expression, forms abnormally in the absence of MEGF7. Digit formation results from complex and coordinated interactions between several signaling molecules 76. These include fibroblast growth factors (FGF), bone morphogenetic proteins (BMP), Wnts (Wnt), and sonic hedgehog (Shh). Loss of MEGF7/LRP4 results in abnormal expression of several of these signaling proteins (FGF8, Shh, BMP2, BMP4, and Wnt7a), as well as an abnormal expression of the Wnt‐ and BMP‐responsive transcription factors Lmx1b and Msx1 75. Moreover the authors show that MEGF7/LRP4 inhibited the Wnt‐induced activation of the luciferase reporter in a Wnt activity assay, suggesting that MEGF7/LRP4 can antagonize the LRP5/6‐mediated activation of this signaling pathway. These findings suggest a role for MEGF7/LRP4 as a modulator of the signaling pathways that control limb development in the embryo.

More recently, Simon‐Chazottes et al. 77 describe two new, independent, allelic mutations at the LRP4 locus resulting in the same phenotype of polysyndactyly as LRP4 ‐/‐. The first allele (digitations anormale—symbol dan) was discovered in a stock of 129S2/SvPas transgenic mice derived from embryonic stem (ES) cells with multiple insertions of a defective strain of Moloney retrovirus (MPSV^{mos‐1 neo}) and was demonstrated by positional cloning to be the consequence of the insertion of a proviral copy into the LRP4 gene. The second allele (malformed digit—symbol mdig) arose spontaneously in the DBA/1LacJ production colony of the Jackson Laboratory. These two recessive mutations of the mouse that cause polysyndactyly confirmed that the gene encoding MEGF7/LRP4 plays an essential role in the process of digit differentiation.

Duchesne et al. 78 have studied syndactyly in Holstein cattle and they could show that MEGF7/LRP4 is most probably also implicated in a bovine limb abnormality. Polysyndactyly is relatively common in human. However, the genetic defect responsible for the malformation has not yet been identified. The aforementioned studies revealed LRP4 as a candidate gene and partly deciphered the complex molecular interactions that take place at the AER during digit formation.

Finally, Tian et al. 79 suggest a postsynaptic role for LRP4. In their study they showed the presence of LRP4 in the brain and demonstrated that LRP4 has postsynapse‐targeting ability. This is possible through the interaction of LRP4 with postsynaptic scaffold proteins, in particular postsynaptic density protein 95 (PSD‐95), and its regulation by calmodulin‐dependent protein kinase II (CAMKII) phosphorylation.

In summary, MEGF7/LRP4 plays a pivotal role in the regulation of essential signaling pathways. MEGF7/LRP4 functions as a modulator of the signaling pathways (Wnt signaling) that control limb development in the embryo and also appears to be involved in various other physiological functions such as brain development.

Conclusion

The LDL‐R gene family constitutes a class of structurally closely related cell surface receptors that fulfill diverse biological functions in different organs, tissues and cell types. Beside endocytosis, considerable evidence has accumulated that shows that the members of the LDL receptor gene family also have fundamental functions in transmitting signals between cells in many, if not all, multicellular organisms. Clearly much work remains on the road toward a comprehensive understanding of the complex biological roles of the members of this family. Identification of the underlying molecular mechanisms may serve to identify new targets for the treatment of different cardiovascular and neuronal disorders and may provide novel insights into embryonic and brain development.