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yeast: endocytosis from the cell-surface, two distinct

biosynthetic pathways, the carboxypeptidase Y (CPY)

and alkaline phosphatase (ALP) from the Golgi,

multivesicular body (MVB) sorting, the cytoplasm to

vacuole pathway (Cvt), macro-autophagy, micro-auto-

phagy, and vacuole inheritance during cell division

(Figure 3A). The endocytic pathway is essential for

regulating levels of cell-surface proteins and intersects

with the CPY pathway at a late endosomal compart-

ment. Components of the CPY-sorting pathway are

directly involved in vacuolar biogenesis, morphology,

and function, as discussed here in greater detail. ALP

travels from the Golgi to the vacuole along a pathway

that is independent of the CPY and endocytic pathways.

Proteins such as aminopeptidase I (API) are delivered

from the cytoplasm to the vacuole through pathways

involving products of the CVT and APG (autophagy)

genes. In micro-autophagy, the vacuole membrane

invaginates to engulf cytoplasmic material, including

entire organelles such as peroxisomes. Vacuolar segre-

gation into dividing daughter cells of budding yeast

requires VAC (vacuolar inheritance) genes, some of

which also participate in the CPY pathway. Previous

studies have also identiﬁed several distinct factors

involved in vacuolar inheritance.

THE CPY PATHWAY

CPY is the prototype of a subset of proteins that

trafﬁcs from the Golgi to the vacuole via an endosomal

intermediate. This pathway depends on the function of

over 60 VPS gene products, mutations in which result

in the mis-sorting of CPY to the secretory pathway,

abnormal vacuole morphology, and in some cases,

abnormal endosome morphology. In a manner analo-

gous to receptor-mediated sorting in mammalian cells,

the CPY receptor Vps10 binds CPY at the Golgi via a

targeting signal. Sorting of this receptor–ligand com-

plex into vesicles bound to endosomes requires

proteins such as clathrin and accessory factors such

as the AP-1 clathrin adaptor complex and the Gga

proteins. Next, the class D Vps proteins are involved in

CPY vesicle targeting and fusion with endosomes, such

as Vps21 (Rab5 homologue), Vps9 (Rab GEF), Vac1

(EEA1), Vps45 (Sec1 homologue), Pep12 (t-SNARE),

and Sec18 (NSF). At the endosome, Vps10 dissociates

from CPY and recycles to the Golgi in a process

requiring the retromer complex (consisting of Vps29,

Vps26, Vps35, Vps5, and Vps17). Mutants defective in

retromer function mis-localize Vps10 to the vacuole

membrane and secrete CPY, as Vps10 becomes limiting

for subsequent sorting reactions at the Golgi.

Another set of Vps proteins (the class E Vps proteins)

is necessary for efﬁcient sorting at the endosome. Class E

Vps mutant cells accumulate abnormal/aberrant endo-

somes containing both biosynthetic cargo such as CPY

and endocytosed proteins. The class E proteins are also

involved in the formation of multivesicular bodies

(MVBs) at late endosomes. The abnormal/exaggerated

endosomes observed in these mutant cells form in part

due to impaired MVB vesicle budding into the lumen of

the endosome.

Finally, fusion of late endosomes/MVBs with the

vacuole requires another set of proteins including Ypt7

(Rab7 homologue) and SNARE proteins (Vti1, Ykt6,

Nyv1, Vam7, and Vam3). The class C Vps protein

complex (also termed HOPS) consisting of Vps18,

Vps11, Vps16, and Vps33 (Sec1 homologue), Vps41,

and Vps39 (Rab GEF) is required for this ﬁnal fusion

step as well (Figure 3B). The Vps34 PI 3-kinase also

contributes to this fusion step by the generation of

PI(3)P that recruits effector proteins such as the PX

domain-containing protein Vam7. Mutants with defects

in these gene products accumulate numerous endoso-

mal intermediates and MVBs that fail to fuse with the

vacuole. Accordingly, mutants defective in components

of the vacuolar fusion machinery display highly

fragmented vacuoles and can sometimes even lack

vacuoles entirely.

THE ALP PATHWAY

ALP is an integral membrane protein that travels from

the Golgi to the vacuole independently of endosomal

compartments that transport CPYand endocytic cargoes

(Figure 3A). A speciﬁc adaptor complex, termed AP-3,

mediates sorting of ALP into vesicles at the Golgi.

However, following formation, fusion of ALP cargo

vesicles with the vacuole is dependent on the class C Vps

complex, Ypt7, Vam7, and Vam3. Thus, the ALP and

CPY pathways converge upon common docking/fusion

machinery at the vacuole (Figure 3B).

CYTOPLASM TO VACUOLE TRANSPORT

AND

MACRO-AUTOPHAGY

Autophagy is a trafﬁcking pathway to vacuoles

regulated by changes in nutrient availability.

In macro-autophagy, induced during starvation, cyto-

plasmic material is ﬁrst sequestered in double-mem-

brane vesicles called autophagosomes and then

subsequently delivered to the vacuole (Figure 3A).

While autophagy is induced, the Cvt pathway con-

stitutively packages the hydrolases aminopeptidase I

(API) and

-mannosidase into autophagosomes for

delivery to vacuoles. Autophagosomes are targeted to,

and fuse with, the vacuole by the same machinery that

mediates endosome-vacuole fusion (Figure 3B). Inside

the vacuole, the lipase Cvt17 is responsible for auto-

phagosome turnover (Figure 2).

VACUOLES 333

FIGURE 3 (A) Model representation of the transport pathways to the vacuole. Endocytosis of cell-surface proteins is followed by transport to

early endosomes and then late endosomes. The CPY pathway sorts proteins from the late Golgi to the late endosome. At the late endosome,

select cargo is sequestered into intralumenal vesicles to form multivesicular bodies (MVB) while other proteins, such as sorting receptors, are

recycled to the Golgi. The ALP pathway functions as a Golgi to vacuole route that is independent of the late endosome/MVB. In the Cvt

pathway, cytoplasmic proteins are enclosed within double membrane structures that subsequently fuse with and deliver their material into the

334 VACUOLES

Vacuole/Lysosome Functions

in Higher Eukaryotes

THE CONTRACTILE VACUOLE

PROTOZOA

Protozoa living in fresh water exist in a hypotonic

environment. Water ﬂows across their plasma mem-

brane since their cytosol is hypertonic to the environ-

ment. To adapt, many protozoa have an organelle, the

contractile vacuole complex (CVC), which collects and

expels excess water. Previous work shows that CVCs are

composed of a central vacuole with radial arm-like

extensions. The extensions are often divided into

separate bundles of tubules and contain proton-translo-

cating V-ATPase enzymes that provide an electrochemi-

cal gradient necessary for ﬂuid collection. The

membrane of the central compartment lacks V-ATPases,

but can expand into a reservoir for ﬂuid storage, and is

capable of fusing with the plasma membrane. It is then

that the central compartment undergoes contraction,

resulting in ﬂuid expulsion.

LYSOSOME-RELATED ORGANELLES

The mammalian vacuole-like lysosome also is a site for

delivery of cellular materials targeted for degradation, as

it is the terminal compartment of the endocytic,

phagocytic, and biosynthetic pathways in mammalian

cells. However, the idea that vacuoles/lysosomes are

simply degradative sites has evolved, in part by the

identiﬁcation of lysosome-like compartments that per-

form additional cellular functions such as lysosomes that

secrete their contents after fusion with the plasma

membrane. Many properties of vacuoles/lysosomes are

shared with this group of cell type-speciﬁc lysosome-

related organelles, which include melanosomes (pigment

granules), lytic granules, platelet-dense granules, baso-

phil granules, nuetrophil granules, and dendrocyte MHC

class II compartments (involved in antigen presentation).

However, in addition to lysosomal proteins, these

organelles contain cell type-speciﬁc components that

are responsible for their specialized functions.

PATHOGEN-CONTAINING VACUOLES

The uptake of foreign objects by macrophages often

provides an important line of immune defense. To some

intracellular pathogens (such as certain protozoa)

however, phagocytosis represents an opportunity to

gain protected access within a host cell. Other types

of pathogens, including Salmonella and Shigella, actively

invade host cells by delivering effectors into the host

cell that leads to their direct uptake into intracellular

vacuoles. Regardless of the mode of entry, the

resulting intracellular vacuole that contains the parasite

undergoes a maturation process, involving numerous

membrane-trafﬁcking events, as well as transmembrane

transport of nutrients. Maturation of the vacuole yields a

unique intracellular environment where the parasites are

not only provided with essential nutrients, but are also

protected from destruction by the host.

DISEASES ASSOCIATED WITH

LYSOSOMAL DISORDERS

Several human genetic disorders are associated with

defects in vacuolar/lysosomal function and transport,

such as I-Cell, Tay-Sachs’, Pompe’s, Galactosialdosis,

and Gaucher’s disease. I-Cell disease is manifested by the

inappropriate targeting/transport of multiple lysosomal

enzymes. Cells of these patients become highly vacuo-

lated and contain numerous dense inclusion bodies.

Patients with I-Cell disease display severe clinical defects

including skeletal and neurological defects, delayed

growth and psychomotor development, and often

death before age ﬁve. Abnormalities in lysosome-related

organelles have also been observed in human genetic

diseases such as the Chediak-Higashi and Hermansky-

Pudlak syndromes. The similarity of genes affected in

these lysosomal diseases to genes involved in vacuolar

transport in yeast further demonstrates the importance

of understanding the molecular machinery involved in

the biogenesis and function of vacuoles, lysosomes, and

lysosome-related organelles. Further studies on vacuole

biogenesis will likely shed light on additional aspects of

vesicular transport in the endosomal, lysosomal, and

secretory systems.

FURTHER READING

Allen, R. D. (2000). The contractile vacuole and its membrane

dynamics. Bioessays 22, 1035–1042.

Bowers, W. E. (1998). Christian de Duve and the discovery of

lysosomes and peroxisomes. Trends Cell Biol. 8, 330– 333.

Bryant, N. J., and Stevens, T. H. (1998). Vacuole biogenesis in

Saccharomyces cerevisiae: Protein transport pathways to the yeast

vacuole. Microbiol. Mol. Biol. Rev. 62, 230–247.

Dell’Angelica, E. C., Mullins, C., Caplan, S., and Bonifacino, J. S.

(2000). Lysosome-related organelles. Faseb J. 14, 1265–1278.

Jones, E. W. (2002). Vacuolar proteases and proteolytic artifacts in

Saccharomyces cerevisiae. Methods Enzymol. 351, 127–150.

Knodler, L. A., and Steele-Mortimer, O. (2003). Taking possession:

Biogenesis of the salmonella-containing vacuole. Trafﬁc 4,

587–599.

Klionsky, D. J., Herman, P. K., and Emr, S. D. (1990). The fungal

vacuole: Composition, function, and biogenesis. Microbiol. Rev.

54, 266–292.

Kornfeld, S., and Mellman, I. (1989). The biogenesis of lysosomes.

Annu. Rev. Cell Biol. 5, 483–525.

Mach, L. (2002). Biosynthesis of lysosomal proteinases in health and

disease. Biol. Chem. 383, 751–756.

Mullins, C., and Bonifacino, J. S. (2001). The molecular machinery for

lysosome biogenesis. Bioessays 23, 333–343.

BIOGRAPHY

Scott D. Emr is a Professor of Cellular and Molecular Medicine at the

University of California, San Diego and an investigator of the Howard

Hughes Medical Institute. His lab focuses on deﬁning components of

the core transport machinery as well as the regulatory apparatus that

direct protein and membrane sorting to and from intracellular

organelles, such as vacuoles.

Christopher J. Stefan is a Research Associate of the Howard Hughes

Medical Institute.

336 VACUOLES

Vascular Endothelial Growth

Factor Receptors

Kenneth A. Thomas

Merck Research Laboratories, West Point, Pennsylvania, USA

Vascular endothelial growth factor receptors (VEGFRs) are a

set of three homologous transmembrane receptor tyrosine

kinases that bind vascular endothelial growth factors (VEGFs).

VEGFRs are expressed primarily by endothelial cells lining the

lumen of vascular and lymphatic vessels. VEGF-mediated acti-

vation of these receptors can induce endothelial cell migration

and mitosis promoting the growth of blood vessels, denoted

angiogenesis, and of lymphatic vessels, or lymphangiogenesis.

VEGFR Genes

EVOLUTION

The three VEGFRs are denoted VEGFR-1, VEGFR-2,

and VEGFR-3, or Flt-1, KDR/Flk-1 and Flt-4, respect-

ively. Each VEGFR gene encodes a protein composed of

seven extracellular immunoglobulin (Ig)-like domains, a

short transmembrane-spanning polypeptide and an

intracellular portion containing a tyrosine kinase. The

VEGFRs are most closely related to the hematopoietic

receptor tyrosine kinases c-kit, c-fms and Flt3 and to the

platelet-derived growth factor receptor (PDGFR)-

and

, each of which contain ﬁve extracellular Ig-like

domains and an intracellular tyrosine kinase. The

VEGFR-1, -2, and -3 genes are clustered with the

hematopoietic receptors and PDGFRs on chromosomes

13, 4 and 5, respectively, consistent with divergence

from a common ancestral receptor tyrosine kinase gene.

STRUCTURE

Each of the three VEGFRs is encoded by 30 exons (NCBI

genomic database, http://www.ncbi.nlm.nih. gov/

genome/guide/human). The corresponding exon-coding

regions, in each gene, are of similar size although some of

the introns that separate them vary substantially in length

resulting in total genomic DNAs ranging from , 190 kb

for VEGFR-1 to 47 kb and 46 kb for VEGFR-2 and -3,

respectively. In all three genes, exon 1 encodes

the secretory leader sequence, exons 2–15 span the extra-

cellular region, exon 16 corresponds to the transmem-

brane-spanning polypeptide and exons 17–30 encode the

cytoplasmic region including the tyrosine kinase con-

taining a kinase –insert domain encoded by exon 21.

Gene Expression

Cellular differentiation status and responses to extra-

cellular signals inﬂuence the expression of each of the

VEGFR genes. Multiple transcription factor DNA-

binding site consensus sequences are present within

approximately the ﬁrst 1 kb 5

of the translational start

site. Transcription factor binding sites within the ﬁrst

intron might also either augment or inhibit transcription.

VEGFR-1

Subsets of vascular endothelial cells, monocytes, den-

dritic cell precursors, and some types of smooth muscle

cells express VEGFR-1. The basal promoter and 5

sequences that control VEGFR-1 endothelial cell-selec-

tive expression contain Ets, Sp1, Egr-1, and CRE

transcription factor binding site consensus sequences,

several of which have been shown to be functional. In

addition, a single hypoxic response element is located

within the ﬁrst 1 kb 5

of the transcriptional start site,

consistent with the observation that the transcription of

VEGFR-1 is increased by hypoxia. Transcription is

initiated downstream of a TATA box basal transcription

factor binding site to generate a 7.5–8 kb mRNA.

VEGFR-2

VEGFR-2 is expressed primarily by vascular endothelial

cells although a few other cell types can also express

this receptor. Selective endothelial cell expression

has been mapped to a region within the ﬁrst 150 bp 5

of the transcriptional start site that contains multiple

Sp1, Ap-2, and NF-

B sites. In vivo expression of

the corresponding murine VEGFR-2 gene has been

studied using transgenic mice. Blood vessel targeted

gene expression in developing mouse embryos is

controlled by sequences not only in the region 5

of the

translational start site but also within the ﬁrst intron. The

human gene, which does not contain a TATA box, is

transcribed as a 7 kb mRNA.

VEGFR-3

Several consensus transcription factor sequences have

also been recognized in the VEGFR-3 gene 5

of the

translational initiation site. A 1.6 kb 5

region has been

reported to drive lymphatic expression. Full-length

VEGFR-3 mRNA is transcribed as a 5.8 kb mRNA.

Protein Structure

EXTRACELLULAR DOMAINS

The primary translation products of the VEGFR-1, -2,

and -3 genes are 1338, 1356, and 1363 amino acid

residue proteins, respectively. Each , 150 kDa protein is

glycosylated at multiple sites on the extracellular Ig-like

domains to generate mature proteins of 185–230 kDa. In

addition, an alternatively spliced soluble 687 amino acid

form of VEGFR-1, denoted sVEGFR-1 or sFlt-1, consists

of the 6 N-terminal Ig-like domains. This 75 kDa protein

is converted to 110 kDa by glycosylation. Partial

proteolysis of VEGFR-3 generates an N-terminal

70 kDa polypeptide disulﬁde linked to the remaining

120–125 kDa transmembrane protein. A crystal struc-

ture of the VEGFR-1 Ig-like domain 2, composed of

5- and 3-stranded

-sheets, in complex with the dimeric

VEGF-A ligand is shown in Figure 1.

INTRACELLULAR DOMAINS

The structure of the VEGFR-2 tyrosine kinase, shown in

Figure 2, contains N- and C-terminal domains consisting

primarily of

-strands and

-helices, respectively. The

catalytic site is located at the interface of these two

domains denoted by the ADP modeled into the structure.

It is ﬂanked by the disordered activation-binding loop,

the glycine-rich nucleotide-binding loop, the catalytic

loop and that can participate in enzyme activation and

substrate binding. The VEGFRs each contain a 65–70

amino acid kinase–insert domain within the N-terminal

region of the C-terminal domain that is deleted in the

crystallized VEGFR-2 kinase. A C-terminally truncated

form of VEGFR-3, missing 65 amino residues, is also

generated by alternative splicing.

Ligand Binding

VEGF STRUCTURE AND

RECEPTOR BINDING

The VEGFs are a family of ﬁve homologous dimeric

glycoproteins. In addition, several VEGF homologues

are expressed by the orf and pseudocowpox viruses,

collectively denoted VEGF-E. HIV tat can also function

as a VEGF. Each ligand binds with pM afﬁnity either 1 or

FIGURE 1 Structure of the complex between a VEGF-A dimer and domain 2 of VEGFR-1. The anti-parallel VEGF-A subunits are red and

blue and the VEGFR-1 domains are green with disulﬁde bonds shown in yellow. Secondary structure is illustrated as arrows pointing toward the

C-terminal ends. (Reproduced from Harada, S., and Thomas, K. A. (2002). Vascular endothelial growth factors. In Principles of Bone Biology

(J. P. Bilezikian, L. G. Raisz and G. A. Rodan, eds.) 2nd edition, Vol 2, pp. 883–902. Academic Press, San Diego, with permission.)

338 VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTORS

2 of the 3 receptors as shown in Figure 3. The core

receptor-binding region of each subunit consists of

, 120 amino acid residues. VEGF subunits, each

composed of four

-strands and two short helices,

dimerize in an anti-parallel arrangement as illustrated by

VEGF-A in Figure 1. The loops between

-strands of

both VEGF-A subunits interact with the second and

third Ig-like domains of each of two receptor subunits

through primarily hydrophobic interactions. Therefore,

VEGF binding can promote receptor dimerization,

which is additionally stabilized by interactions between

the fourth Ig-like domains in VEGFR-1, as illustrated in

Figure 4. VEGF-C and -D bind with highest afﬁnity to

VEGFR-3, mainly expressed on lymphatic endothelial

cells and on the tips of some growing capillaries. Partial

proteolysis, which removes sequences N- and C-term-

inal of the core-receptor-binding region, can increase the

afﬁnity of VEGF-C and -D for VEGFR-2 as indicated by

the dashed lines in Figure 3.

CORECEPTORS

VEGF-A, VEGF-B, and PlGF are subject to alternative

splicing that either incorporates or deletes C-terminal

polycationic regions. These positively charged sequences

can promote binding to negatively charged soluble

heparin and heparin sulfate proteoglycans and to the

membrane-anchored proteins neuropilin-1 and -2.

Although these receptors do not appear to induce

VEGF signal transduction, they might indirectly pro-

mote VEGF activity by partitioning the ligands to

FIGURE 2 Structure of the VEGFR-2 tyrosine kinase. The N- and C-terminal lobes are blue and red, respectively. The active site region

between these two domains is denoted by the location of ADP modeled into it. The glycine-rich, catalytic, and the ends of the disordered activation

loops are shown in yellow. The ends of the largely deleted kinase-insert domain are also shown and labeled. (Reproduced from Harada, S.,

and Thomas, K. A. (2002). Vascular endothelial growth factors. In Principles of Bone Biology (J. P. Bilezikian, L. G. Raisz and G. A. Rodan, eds.)

2nd edition, Vol 2, pp. 883– 902. Academic Press, San Diego, with permission.)

FIGURE 3 Receptor-ligand binding selectivity. VEGFs are listed in

the top row with color-coded arrows pointing toward the high afﬁnity

VEGFRs that they bind. Tat and VEGF-E function as viral VEGFs.

Dashed arrows from VEGF-C and -D to VEGFR-2 indicate high-

afﬁnity binding following full proteolytic processing to remove N- and

C-terminal polypeptides. (Modiﬁed from Harada and Harada, S., and

Thomas, K. A. (2002). Vascular endothelial growth factors. In

Principles of Bone Biology (J. P. Bilezikian, L. G. Raisz and G. A.

Rodan, eds.) 2nd edition, Vol 2, pp. 883–902. Academic Press, San

Diego, with permission.)

VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTORS 339

cellular surfaces, thereby increasing their local concen-

tration in the vicinity of the high-afﬁnity receptors, and

by functioning as co-receptors that present the ligands to

the high-afﬁnity VEGFRs.

Signal Transduction

RECEPTOR ACTIVATION

Intracellular signaling is initiated by VEGF-induced

receptor dimerization that brings the intracellular

tyrosine kinases into proximity where they are thought

to phosphorylate each other as shown in Figure 4. The

initial phosphorylation of tyrosine residues 1054

and 1059 on the tyrosine kinase activation loop of

VEGFR-2 decreases the K

of the enzyme for ATP and for

peptide substrates without altering K

cat

, the effective

maximal rate of enzyme activity. This increased afﬁnity

for substrates could reﬂect the movement of the

phosphorylated activation loop to provide better sub-

strate access to their binding sites. Equivalent activation

loop tyrosines exist on VEGFR-1 and -3 so that they

might function in a similar manner.

RECRUITMENT OF SIGNALING PROTEINS

Additional tyrosines in the juxtamembrane region, the

kinase–insert domain and the C-terminal tail can be

phosphorylated, as shown schematically in Figure 4.

Some of these phosphorylated tyrosine residues have

been shown to serve as recognition sequences for binding

by one or more signal transduction proteins. These

include adapter proteins such as Grb2, Grap, Nck, Crk,

Sck, Shc, and Vrap, which can bridge the receptors to

other phosphorylated and non-phosphorylated proteins,

and several enzymes such as phospholipase C

(PLC

phosphatidylinositol 3-kinase (PI3K), and the tyrosine

phosphatase SHP-2. VEGF-induced phosphorylation is

rapid, reaching maximal levels by 2-5 min, followed

within 30 min by receptor internalization.

Some of these phosphorylated tyrosine residues have

been linked with speciﬁc functions. For example,

phosphorylation of VEGFR-2 Tyr

1175

in the C-terminal

tail is required for the efﬁcient phosphorylation of

PLC

and mitogenic activity. The longer alternatively

spliced form of VEGFR-3 contains tyrosines within

the unique C-terminal 65 amino acid residues. At least

one of these tyrosines, Tyr

1337

, is required for trans-

forming activity in vitro and upon phosphorylation

can bind the adapter protein Shc.

SIGNAL TRANSDUCTION CASCADES

The phosphorylation of VEGFR-2-associated PLC

activates its enzymatic activity catalyzing the hydro-

lysis of membrane-anchored phosphatidylinositol

4,5-bisphosphate (PIP

) to generate inositol 1,4,5-

trisphosphate (IP

) and diacylglycerol (DAG). Water

soluble IP

activates an endoplasmic reticulum Ca

2þ

channel that releases Ca

2þ

, activating several enzymes

including endothelial cell nitric oxide synthase (eNOS)

and promoting the translocation of speciﬁc protein

kinase C (PKC) isoforms to the membrane where it is

activated by binding membrane-associated DAG. PKC

appears to be able to activate the mitogen-activated

protein kinase kinase (MAPKK) mitogenic pathway

possibly involving Raf kinase. In addition, nitric oxide

can activate protein kinase G (PKG) that subsequently

activates Raf. MAPKK activates mitogen activated

FIGURE 4 The structure of VEGFRs, containing seven extracellular Ig-like domains, a juxtamembrane region, the two domain tyrosine kinase

containing a kinase-insert domain and a C-terminal tail, is shown on the left. Binding of dimeric VEGF to Ig domains 2 and 3 induce receptor

dimerization, which can be aided by interactions between receptor domains 4. This promotes phosphorylation of two kinase activation loop

tyrosines, denoted by the red “P” labels, and enzymatic activation in VEGFR-2 and probably in VEGFR-1 and -3. Additional tyrosines in the

juxtamembrane region, kinase-insert loop and C-terminal tail are phosphorylated, as denoted by blue “P” labels, and bind signaling proteins that

trigger several VEGF functions.

340 VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTORS

kinase (MAPK), which can enter the nucleus to

modulate transcription. Additional pathways,

mediated through PI3K and the anti-apoptotic kinase

Akt, promote cell adhesion, migration, and survival.

Biologic Activities

DEVELOPMENT

The developmental activities of VEGFRs have been

revealed by mouse gene knockouts, which are each

embryonically lethal. Blood vessels exist in VEGFR-1

knockout mice but are disorganized, which appears to be

a consequence of modiﬁed cell fate leading to increased

numbers of endothelial cell progenitor hemangioblasts

that altered vessel pattern formation. Surprisingly,

knockout of the VEGFR-1 gene segment encoding the

tyrosine kinase but retention of the extracellular and

membrane-spanning regions leads to normal develop-

ment of blood vessels and survival. This result is

consistent with the possibility that one of the functions

of VEGFR-1, which binds VEGF-Awith , 10-fold higher

afﬁnity than VEGFR-2, might be to sequester low levels

of VEGF, thereby “buffering” the VEGFR-2 receptor

from inappropriate activation by low levels of VEGF.

The VEGFR-1 cytoplasmic region might inhibit the

VEGFR-2 mitogenic, but not migratory, activity by a

mechanism involving VEGFR-1 juxtamembrane Tyr

794

The VEGFR-2 knockout is virtually devoid of vascular

endothelial cells, consistent with its crucial role as a

mediator of endothelial cell mitosis and survival.

VEGFR-3 gene knockout mice exhibit vasculogenesis

and angiogenesis in early developing embryos but large

vessels appear abnormal with defective lumens leading

to ﬂuid accumulation in the pericardial cavity and

cardiovascular failure. Therefore, at least during early

embyrogenesis before lymphatic vessels develop from

post-capillary venules, VEGFR-3 plays a critical role in

vascular development.

ANGIOGENESIS

In adults, VEGFR-2 is the primary angiogenic receptor

family member. It can mediate the migration and mitosis

of vascular endothelial cells culminating in neovascular

growth. Ligands such as VEGF-A and the viral VEGFs

that bind VEGFR-2 are sufﬁcient to drive angiogenesis

and the growth of some hematopoietic progenitor cells.

Antibodies to VEGFR-2 and enzyme inhibitors of the

VEGFR-2 tyrosine kinase inhibit angiogenesis and the

resulting growth of tumors in mice.

Soluble VEGFR-1 (sVEGFR-1), which is expressed

by cultured endothelial cells and has been detected

in vivo, is the only identiﬁed naturally occurring

speciﬁc VEGFR inhibitor. It retains the ligand-binding

site and so recognizes the same VEGFs as full-

length VEGFR-1. The soluble receptor can not only

homodimerize but also heterodimerize with the extra-

cellular regions of VEGFR-1 and VEGFR-2. Therefore,

it can sequester ligands and perhaps inhibit activation

of full-length membrane-spanning VEGFR-1 and

VEGFR-2 by the formation of dominant negative

heterodimers that contain a single tyrosine kinase so

are incapable of kinase activation by trans-phosphoryl-

ation. Transfection experiments show that expression

of sVEGFR-1 by tumor cells that express transfected

sVEGFR-1 exhibit severely inhibited growth in vivo

but not in vitro, consistent with an antiangiogenic

mechanism.

LYMPHANGIOGENESIS

VEGFR-3 is expressed by lymphatic endothelial cells

and also vascular endothelial cells at the tips of growing

capillary shoots. The VEGFR-3 ligand VEGF-C is

mitogenic for lymphatic endothelial cells in culture.

Transgenic mice expressing elevated VEGF-C or -D in

skin induce the growth of dermal lymphatic vessels but

not blood vessels. However, inhibitory anti-VEGFR-3

antibodies can inhibit tumor angiogenesis. Naturally

occurring human missense mutations with inactive

tyrosine kinases are linked to lymphoedema, a genetic

disease in which ﬂuid accumulates in tissues as a

consequence of deﬁcient lymphatic function. Therefore,

in adults VEGFR-3 appears to be active on lymphatic

endothelial cells and at least some endothelial cells in

actively growing capillaries.

NONMITOGENIC FUNCTIONS

In adults, activation of VEGFR-2 induces vascular

permeability that can facilitate angiogenesis and pro-

mote edema. Although VEGFR-1 does not directly

promote permeability, it might play a permissive role.

Activation of VEGFR-1 by its selective ligands PlGF and

VEGF-B does not appear to directly induce endothelial

cell mitosis or angiogenesis under most conditions.

However, it can induce vascular endothelial cell

expression of speciﬁc proteins such as tissue factor,

matrix metalloproteases, urokinase, plasminogen acti-

vator inhibitor-1, hepatocyte growth factor and pigment

epithelium-derived factor. In addition, activation of

monocyte and macrophage VEGFR-1 can drive

expression of tissue factor, monocyte chemoattractant

protein-1 and tumor necrosis factor-

. It can also induce

the production of endothelial cell nitric oxide, and the

migration of monocytes and some endothelial cells.

Although the VEGFR-1 ligand PlGF is not a potent

angiogenic agent, it does appear to promote the

development of collateral blood vessels, a process

involving the conversion of smaller to larger vessels.

VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTORS 341

Therefore, on the basis of the knockout and cell

expression data, VEGFR-1 seems to modulate the

expression of several genes associated with differen-

tiated functions and the activity of VEGFR-2.

Summary

The homologous VEGFRs are critical mediators of the

growth and function of blood and lymphatic vessels.

These receptors and their ligands are intimately involved

not only in development but also in the maintenance of

normal differentiated functions. Their inappropriate

activation or inhibition can have pathologic conse-

quences so they also are potential targets for therapeutic

intervention.