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Vasopressin/Oxytocin

Receptor Family

Michael J. Brownstein

National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, USA

Large neurons at the base of the brain (the hypothalamus) of

humans and other mammals send their axons through the

median eminence to terminate in the posterior lobe (neurohy-

pophysis) of the pituitary. Each of these “magnocellular” nerve

cells typically synthesizes one of two neurohypophyseal

hormones – vasopressin (VP) or oxytocin (OT). In fact, all

vertebrates higher than jawless ﬁsh make vasopressin- and

oxytocin-like hormones. Jawless ﬁsh and invertebrates, on the

other hand, appear to produce only one VP/OT-like peptide

per species.

The Structures of “Pressins”

and “Tocins”

For the last billion years, the peptides in VP/OT family

have retained certain structural features. With the

exception of the hydrins – partially processed relatives

of vasotocin, which are physiologically active in some

amphibia – they all have nine amino acids and a

C-terminal amide. Cysteine (C) is invariably present in

positions 1 and 6, and the two cysteine residues form a

cystine bridge, creating conformationally constrained,

cyclic peptides. Except for seritocin (serine5, isoleu-

cine8-oxytocin), which was reported in a single species,

Bufo regularis, all family members have asparagines (N)

in position 5, and proline (P) and glycine (G) in positions

7 and 9, respectively. VP-like peptides (“pressins”) are

characterized by the presence of arginine (R) or lysine

(K) in position 8, while OT-like molecules (“tocins”)

have leucine (L), isoleucine (I), glutamine (Q), valine

(V), or threonine (T) there. All of the invertebrate

peptides except for the locust diuretic hormone, which

has not been detected in other insect species, have an R

instead of Q, N, or S in position 4. Thus, the primordial

peptide that gave rise to the molecules we know today

should have looked rather similar to one of the

conopressins or to vasotocin, the likely ancestor of all

of the vertebrate hormones. (The molecules detected

in Hydra with antivasopressin antibodies share the

C-terminal PRG-amide motif with VP, but are

otherwise unrelated.)

In addition to the peptides shown in Table I and

Figure 1, it is worth noting that the major metabolite of

VP (pGlu4, Cystine6-AVP) mobilizes calcium in some

cells in the central nervous system, and has been

suggested to have a unique, but as yet unidentiﬁed,

receptor of its own.

Peptide Biosynthesis

The peptides in the VP/OT family are synthesized as

parts of precursor proteins. The vasopressin precursor

has the following structural features:

signal peptide-CYFNQNCPRGG-K

neurophysin-copeptin

The signal peptide is removed cotranslationally in the

endoplasmic reticulum, liberating the N terminus of the

peptide. Then, after the precursor is packaged into

vesicles by the Golgi apparatus, it is cleaved at the

asterisked sites by a Lys-Arg calcium-dependent endo-

protease. The remaining basic amino acids are

removed by a carboxypeptidase B-like enzyme, leaving

a glycine (G) on the carboxy terminus. This glycine

serves as the donor of the amide group, which is

generated by the sequential actions of a peptidyl-glycine

monooxygenase and a peptidyl-hydroxyglycine lyase.

Neurophysin is thought to serve as an internal chapar-

one, promoting proper folding and cystine-bridge

formation. Copeptin, a glycosylated species, is part of

the vasopressin, but not the oxytocin precursor. Its

function is unknown. The genes encoding the VP and

OT precursors are adjacent to one another on the same

chromosome. In mammals, they are oriented tail-to-tail,

and share regulatory elements.

The Functions of Vasopressin

and Oxytocin

VP/OT-like peptides play different roles in the

various species where they are found. They are very

commonly involved in water homeostasis and/or

reproductive function. In humans vasopressin is an

antidiuretic hormone. It is released from the posterior

pituitary into the blood stream when the osmolarity

(solute content) of the blood increases, and it acts on V2

receptors in the kidney to cause water retention. V2

receptors are Gs-coupled, and increase intracellular

cyclic AMP. This, in turn, causes preformed aquaporin

(AQP) 2 channels to be inserted into the luminal surfaces

of cells in the collecting duct of the kidney. Water enters

these channels, traverses the cells, exits through AQP 3

and 4 channels on their interstitial faces, and enters

the bloodstream. Mutations in the genes encoding

vasopressin, the V2 receptor, or AQP2 cause diabetes

insipidus – an inability to concentrate urine, resulting in

high urine volumes and a need to consume large amounts

of ﬂuid each day. Since the V2 receptor gene is on the X

chromosome, female carriers pass X-linked nephrogenic

diabetes insipidus along to their sons, but not their

daughters.

Nonosmotic as well as osmotic stimuli can trigger

vasopressin secretion. Osmoreceptors on cells in the

anterior hypothalamus, and baroreceptors in the left

ventricle of the heart, aortic arch, and carotid

sinus monitor osmolarity and blood pressure, respect-

ively, and participate in controlling the ﬁring of

VP-producing cells.

In addition to V2 receptors, vasopressin acts on V1a

and V1b receptors. The former are found on blood

vessels and are responsible for VP’s pressor (blood

pressure increasing) activity. The physiological impor-

tance of this is moot, but it is clear that certain

vascular beds are especially sensitive to VP, including

those in the skin and uterus. In fact, it has been

suggested that V1a receptor antagonists might be

useful for treating Raynaud’s disease (excessive constric-

tion of digital arteries) and dysmenorrhea (menstrual

cramps which may be caused by dilation of vessels in

the uterus).

V1b receptors are found on corticotrophs (adreno-

corticotropic hormone or ACTH-producing cells) in

the anterior pituitary. In addition to being made by

magnocellular neurons, VP is also synthesized by small

cells in the paraventricular nucleus of the hypothala-

mus. These same cells also make corticotropin releas-

ing hormone (CRH), and their axons terminate on

portal vessels in the external zone of the median

eminence. VP and CRH are released into these vessels

and transported to the anterior pituitary, where they

act in concert to stimulate ACTH secretion. ACTH, in

turn, causes the adrenal to release glucocorticoids.

Thus, VP is important in mediating the body’s

response to stress.

TABLE I

Vasopressin, Oxytocin, and Related Peptides

Oxytocin-like peptides

(Tocins)

Oxytocin (OT) C YIQNCP LG-amide Mammals, Paciﬁc ratﬁsh

Mesotocin (MT) CYIQNCPIG-amide Nonmammalian tetrapods, marsupials, lungﬁsh

[Phe2]Mesotocin (FMT) CFIQNCPIG-amide Australian lungﬁsh

Seritocin (ST) C YIQSCP IG-amide Dryness-resistant African toad

Isotocin (IT) C YISNCP IG-amide Bony ﬁsh

Aspargtocin (AspT) C YINNCP LG-amide Spiny dogﬁsh

Asvatocin (AsvT) C YINNCPVG-amide Spotted dogﬁsh

Glumitocin (GT) C YISNCPQG-amide Rays

Phasvatocin (PhT) C YFNNCPVG-amide Spotted dogﬁsh

Valitocin (ValT) C YIQNCPVG-amide Spiny dogﬁsh

Annetocin (AnT) C FVRNCPTG-amide Earthworm

Cephalotocin (CT) C YFRNCP IG-amide Octopus

Vasopressin-like peptides

(Pressins)

A-Vasopressin (AVP) C YFQNCPRG-amide Most mammals

L-Vasopressin (LVP) C YFQNCPKG-amide Pig, marsupials

Phenypressin (PP) C YFQNCPRG-amide Marsupials

Vasotocin (VT) C YIQNCPRG-amide Non-mammalian vertebrates

Hydrin 2 (H2) CYIQNCP RGG Frogs, toads

A-conopressin (ACP) C IIRNCP RG-amide Snail

L-conopressin (LCP) C FIRNCP KG-amide Snail, sea hare, leech

Locust diuretic C LITNCP RG-amide Locust (but not fruitﬂy)

hormone (LDH)

Putative Ancestor C(F/Y)I(Q/R)NCP(R/K)G-amide

Abbreviations: C (cysteine), F (phenylalanine), G (glycine), I (isoleucine), K (lysine), N (asparagine), P (proline),

Q (glutamine), R (arginine), S (serine), T (threonine).

344 VASOPRESSIN/OXYTOCIN RECEPTOR FAMILY

V1a and V1b, but not V2, receptors are found in

the brain. The central effects of vasopressin agonists

and antagonists must be mediated by these receptors.

These effects vary from species to species, and they

are unknown in humans. It appears, however, that VP

may mediate behavioral reactions to stress, aggressive

and afﬁliative behavior, juvenile recognition, and

parenting in rodents. In addition, V1a antagonists

appear to block the vomiting associated with

motion sickness in pigs and ferrets. This appears to

be a central effect of the drugs, but could have a

peripheral component.

Oxytocin-producing mammals make a single recep-

tor for this peptide. Like the V1a and V1b receptors, it

is Gq-coupled, and activates phospholipase C, increas-

ing intracellular calcium. Oxytocin receptors increase

dramatically in the pregnant uterus as term approaches.

Despite the fact that it has been used for decades to

induce uterine contractions, oxytocin is not essential for

this process, and oxytocin receptor antagonists have not

Jawless fish

Cartilaginous fish

Bony fish

Amphibians

Lizards, birds

Egg-laying mammals

Marsupials

Placental mammals

Pig

Rat, mouse

Man

1000

560

530

450

360

310

(>170?)

170

1 peptide

per species

2–3 peptides

per species

Arthropods, nematodes, molluscs(?)

Pressins Tocins

ACP, LCP

LDH

AnT, CT

AspT, AsvT, GT

PhT, ValT, OT

VT IT, MT, FMT

VT, H2 MT, (ST)

VT MT

AVP OT

LVP OT

AVP OT

AVP, LVP

}

FIGURE 1 Evolution of vasopressin-related (pressins) and oxytocin-related (tocins) peptides. The timescale (millions of years ago) was taken

from Hedges (2002). Note that invertebrates and jawless ﬁsh make a single pressin or tocin per species, and that vertebrates make two or, in the case

of marsupials, three such peptides – one or two pressins and one tocin. It appears that vasotocin was the ancestor of the vertebrate hormones, and it

continued to be synthesized by all vertebrates except mammals, in which it was replaced by arginine or lysine vasopressin. Tocins began to be

produced in ﬁsh. It is not clear why cartilaginous ﬁsh make so many of these; lungﬁsh make MTor FMT, and all of the remaining bony ﬁsh make IT.

Based on the structures of the peptide precursors, it has been argued that lungﬁsh are related to tetrapods (e.g., toads) and that bony ﬁsh form a

separate lineage. Amphibians, lizards, birds, and marsupials make MT; egg-laying and placental mammals differ in the hormones they produce.

Marsupials appear to have branched away as suggested by other analyses. The abbreviations used in this ﬁgure are deﬁned in Table I.

VASOPRESSIN/OXYTOCIN RECEPTOR FAMILY 345

Human V1B

Tree frog MT

Human OT

Human V1A

White sucker IT

Flounder AVT

Chicken VT1

Human V2

Snail CP1

Snail CP2

GRDEE LAKV E I GVL A TV LV LATGGNL AVL L T LGQLGRK - - RSRMHL FV LHL A L TDL AVAL

KRNEEIAKVEVTVLALILFLALAGNICVLLGIYINRHK- -HSRMYFFMKHLSIADLVVAV

RRNEALARVEVAVLCLI LLLAL SGNACVLLALRTTRQK - -HSRLFFFMKHLS IADLVVAV

VRNEELAKLEIAVLAVTFAVAVLGNSSVLLALHRTPRK- - TSRMHLFI RHLSLADLAVAF

GRNEEVAKMEITVLSVTFFVAVIGNLSVLLAMHNTKKK- - SSRMHLFIKHLSLADMVVAF

GRNEEVAQI EIMVLSI TFVVAVI GNVSVLLAMYNTKKK- -MSRMHL FIKHLSLADLVVAF

ERDEQLAQVEIAVLGVI FLTASVGNF I L I LVLWRRRKK- - LSRMYVFMLHLS IADLVVAF

TRDPLLARAELALLSIVFVAVALSNGLVLAALARRGRRGHWAPIHVFIGHLCLADLAVAL

GVDEDLLKIEIAVQATILYMTLFGNGIVLLVLRLRRQK- -LTRMQWFIAHLAFADIFVGF

GRDESLAR I EI LVQSI I LALA I I GNSCVLTALARRGKA - - ASRMHL F I FHLS I ADLLVAV

TM1

TM2

Human V1B

Tree frog MT

Human OT

Human V1A

White sucker IT

Flounder AVT

Chicken VT1

Human V2

Snail CP1

Snail CP2

FQVLPQLLWDI TYRFQGPDLLCRAVKYLQVLSMFASTYMLLAMTLDRYLAVCHPLRSLQQ

FQVLPQL IWDITFRFYAPDFVCRLVTYLQVVGMFASTYMLL LMSLDRCLA ICQPLRSLHR

FQVLPQLLWDI TFRFYGPDL LCRLVKYLQVVGMFASTYLLLLMSLDRCLA ICQPLRSLRR

FQVLPQMCWDI TYRFRGPDWLCRVVKHLQVFGMFASAYMLVVMTADRYI AVCHPLKTLQQ

FQVLPQLCWEITFRFYGPDFLCRIVKHLQVLGMFASTYMMVMMTLDRYIAICHPLKTLQQ

FQVLPQLCWE ITYRFFGPDFLCRI VKHLQVTGMFASTYMMVMMTLDRY IA I CHPLKTLQQ

FQVLPQL IWDITDVFI GPDFLCRI IKYLQL LGMFASTYMI VVMTVDRYQAVCYPMVTFQK

FQVLPQLAWKATDRFRGPDALCRAVKYLQMVGMYASSYMI LAMTLDRHRAICRPMLAYRH

FNI LPQL I SDVTI VFHGDDFTCRF IKYFQV I AMYASSYVLVMAAI DRYL SICHPLTSQTL

FNI LPQL IWDITERFYGGDL LCRY I KFMQVYVMYLSTYMLVMTAVDRYRAVCHPLSAFNT

TM3

Q1 Q2

C1 R2

Human V1B

Tree frog MT

Human OT

Human V1A

White sucker IT

Flounder AVT

Chicken VT1

Human V2

Snail CP1

Snail CP2

PG-QSTYLL I AAPWLLAA I FSL PQVFIFSLREV IQGSGVLDCWADFGFPWGPRAYLTWTT

- - -RSDCVYVLFTWI LSFLLS I PQTA I FSLT EVENG- -VYDCRADF I L PWGPKAY I TWI T

- - -RTDRLAVLATWLGCLVASAPQVHIFSLREVADG- -VFDCWAVFIQPWGPKAYI TWIT

PA-RRSRLMIAAAWVLSFVLSTPQYFVFSMIEVNNVTKARDCWATFIQPWGSRAYVTWMT

PT-QRAYIMIGSTWLCSLLLSTPQYFIFSLSEIQNGSYVYDCWGHFIEPWGIRAYITWIT

PT-QRSYIMIVSTWMCSLVFSTPQYFIFSLSEVKNGSTVKDCWAHFIEPWGARAYITWIT

KR-ALWNI PICTSWSI SL I LSL PQVFIFSKI EI SPG- - I FECWAEFIQPWGPRAYVTWIL

GSGAHWNRPV LVAWAFSL LLSL PQL F I FAQRNVEGGSGVTDCWACFAEPWGRRTYVTWI A

SP-KRVHLMIALAWLI SFLCALPQVFIFSLQAVGPD- -QYDCLATFEPDWGMQAYITWVF

STRTPLYCMIVSAYV I SGVLSL PQPI IFKYREKSHGSGDYECWGRFEPPWTLNLY ITSFT

TM4

Human V1B

Tree frog MT

Human OT

Human V1A

White sucker IT

Flounder AVT

Chicken VT1

Human V2

Snail CP1

Snail CP2

LAI FVLPVTMLTACYSL ICHEICKNLKVKTQAWRVGGGG- - - - - - - - - - - - - - - -WRTWD

LSVYIIPVLILSICYGLISYKIWQNIRLKTVCESN-----------------------LR

LAVYIVPVIVLATCYGLISFKIWQNLRLKTAAAAA-----------------------AE

GGIFVAPVVILGTCYGFICYNIWCNVRGKTASRQSKGAE----------------QAGVA

VGIFLIPVIILMICYGFICHSIWKNIKCKTMRGTR------------------------N

GGI FLVPVVI LVMCYGF ICHTIWKNIKYKKRKTI PG- - - - - - - - - - - - - - - - - - - - - -AA

VVI FF I PSTIL I TCQVK ICKI IKRNIYVKKQNEYQ- - - - - - - - - - - - - - - - - - - - - - - -V

LMVFVAPTLGIAACQVLIFREIHASLVPGPSERPG------------------------G

VANYV I PFLLLAFCYGRI CHVVWMSVAAKESAAYSSMRNGCTESSRP- - - - - IKMRI SFH

FAVYI VPLA IL I FAYVSI CCTIWRKYKSAENERKHMLNGSDSSLGNRNI YSNHVTHSALF

TM5

Human V1B

Tree frog MT

Human OT

Human V1A

White sucker IT

Flounder AVT

Chicken VT1

Human V2

Snail CP1

Snail CP2

RPSPSTLAAT--------TRGLPSR----VSSINTISRAKIRTVKMTFVIVLAYIACWAP

LSTSRRAT--------------LSR----VSSVRLISKAKIRTVKMTFIIVLAYIVCWTP

APEGAAAGDG--------GRVALAR----VSSVKLISKAKIRTVKMTFIIVLAFIVCWTP

FQKGFLLA--------------PC-----VSSVKSISRAKIRTVKMTFVIVTAYIVCWAP

TKDGMIGK--------------VS-----VSSVTIISRAKLRTVKMTLVIVLAYIVCWAP

SKNGLIGK--------------NS-----VSSVTTISRAKLRTVKMTFVIVLAYIICWAP

TNQKQVLP---------------SR----ASSVNCISKAMIKTVKMTIVTVVAYVLCWSP

RRRGRRTG---------------S-----PGEGAHVSAAVAKTVRMTLVIVVVYVLCWAP

RRRDNTNATLTSLDRHDASAVTSSDSKKPRGHQRGVSKSKMKTI KL TLTVVLCYL FCWAP

RHRGV I ERRRNLVQRCRPAPMAAPR----AHSLRGFSRAKLKTVKLTFVVIVAYVVCWSP

TM6

Human V1B

Tree frog MT

Human OT

Human V1A

White sucker IT

Flounder AVT

Chicken VT1

Human V2

Snail CP1

Snail CP2

FFSVQMWSVWDKNAPDEDSTNVAFTISMLLGNLNSCCNPWI YMGFNSHL

FFFVQMWSVWDPNPPKE- - -ASLFI IAMLLGSLNSCCNPWIYMLFTGHL

FFFVQMWSVWDANAPKE- - -ASAFI IVMLLASLNSCCNPWIYMLFTGHL

FF I I QMWSVWDPMSVWTESENP T I T I TA LL GSL NSCCNPWI YMF FSGHL

FF I VQMWSVWDENFSWDDSENAAVTLSALLASLNSCCNPWI YML FSGHL

FFTVQMWSVWDENFQYADSENTAVTISALLASLNSCCNPWIYMI FSGHL

FF I AQLWSVWF PSG I TEG- - - SAF T I IML L GNLNSCTNPWI YMYFCGH I

FFLVQLWAAWDPEAPLEG- - -APFVLLMLLASLNSCTNPWI YASFSSSV

FFVVQMWSAFDDDSGI EH- - - PVTVI CMLLASLNSCTNPWIYLAFSGRT

FFL SQLWWLYDEQQEHN- - - -HAVVIMVLLASLNSCCNPWI YLAFSGNL

TM7

346 VASOPRESSIN/OXYTOCIN RECEPTOR FAMILY

proven useful for treating preterm labor. There is no

doubt, however, that the pulsatile release of OT from the

pituitary in response to cervical dilation and vaginal

stimulation, facilitates the expulsion of the fetus.

Oxytocin is required for milk ejection. Mechanical

stimulation of pressure sensitive receptors in the nipple

of the breast by the nursing infant results in activation of

magnocellular neurons in the hypothalamus and release

of pulses of OT into the bloodstream. The hormone

causes breast myoepithelial cells to contract, increasing

intramammary pressure and forcing milk into the ducts.

In the absence of OT, milk cannot be let down, and the

infant will starve if it is not provided an alternative

source of food.

In addition to its roles in parturition and lactation,

oxytocin appears to affect maternal and social beha-

viors, stimulate lipogenesis to compensate for lipid loss

in the milk (via an action on insulin secretion), and

possibly participate in regulating salt and water balance.

While OT causes natriuresis in rats, it is not clear that

this is the case in humans.

Vasopressin and

Oxytocin Receptors

As expected from the fact that their ligands are similar,

VP and OT receptors are structurally related. They are

members of the rhodopsin superfamily, and have seven

a-helical membrane-spanning domains connected to one

another by intracellular and extracellular loops. The N

terminus of each receptor faces the outside of the cell;

the C terminus is cytoplasmic. The intracellular loops

and C-terminal tail of the receptors interact with G

proteins, coupling agonist binding to activation of

second messenger systems. More than 40 VP/OT

receptors found in species ranging from snails to humans

have been cloned and sequenced. The primary sequences

of some of these are shown in Figure 2. It is remarkable

that from the beginning of its ﬁrst transmembrane

domain (TM1) to the end of its seventh one (TM7),

the snail conopressin receptor 2 is 43% identical in

amino acid sequence to the human V1a, V1b, and OT

receptors and the white sucker ﬁsh vasotocin recep-

tor. Unlike the vertebrate proteins, however, the

conopressin receptor responds equally well to lysine8-

and isoleucine8-conopressin (an OT-like synthetic

analogue of lysine-conopressin). Duplication of a rela-

tively promiscuous receptor of this sort might

have permitted trial-and-error evolution of functionally

distinct pressors and tocins in vertebrates.

A number of amino acids are conserved among all of

the receptors in Figure 2. Some of these residues are

found in most G-protein-coupled receptors. Among

them, the arginine (R2) in the DRY motif, found just

beneath TM3, is thought to dwell in a pocket formed by

polar residues in TMs 1, 2, and 7 when the receptor is in

its inactive state. Hormone binding dislodges this

arginine from its polar pocket, exposing G-protein

docking sites on the cytoplasmic loops.

The cysteines in extracellular loops 1 and 2 (C1 and

C2, respectively) are also highly conserved among

rhodopsin-like receptors. They form a cystine bridge

that links these loops, stabilizing the conformation of

the receptors. The pair of cysteines (C3 and its neighbor)

located 15 aa’s below TM7 in the cytoplasmic tails of

most VP/OT receptors are likely to be palmitoylated and

are thought to anchor their C termini to the plasma

membrane. Like other members of the rhodopsin

superfamily, VP and OT receptors appear to be

glycosylated on their N termini, and regulated by

phosphorylation of their intracellular domains.

A number of attempts have been made to model

the binding of VP, OT, and vasotocin to their

receptors. The models are fundamentally similar in

the sense that they all predict that the peptide

hormones ﬁll a cleft located in the upper third of

the barrel formed by the seven membrane-spanning

-helices. The hydrophobic amino acids that comprise

the cyclic portion of the peptides (cysteine1, tyro-

sine2, isoleucine or phenylalanine3, and cystein6)

appear to reside in a hydrophobic pocket formed by

aromatic residues on helices 5 and 6 (and adjacent

helices). The more polar amino acids (asparagine4,

glutamine5), and the amidated C terminus of the

hormones must occupy a hydrophilic region formed

by residues on helices 2, 3, and 4. More speciﬁcally,

residues that are conserved in the N-terminal domain

and TMs 2, 3, 4, and 6 in most, if not all, of the

VP/OT receptors (labeled R1, Q1, Q2, K, Q3, Q4,

and Q5 in Figure 2) have been shown to be important

for high-afﬁnity binding, even though their predicted

interactions with speciﬁc amino acids in the peptide

hormones vary from model to model. Parsimony

dictates that residues conserved among the various

FIGURE 2 Alignment of vasopressin and oxytocin receptors and selected relatives. To save space, the extracellular N termini and intracellular C

termini have been removed. They are quite divergent, but it is remarkable that the transmembrane domains (TMs), the ﬁrst two extracellular loops

(linking TMs 2 and 3, and TMs 4 and 5), and portions of intracellular loops 2 and 3 (linking TMs 3 and 4, and TMs 5 and 6), have remained so

similar throughout the course of their evolution. Speciﬁc residues in these domains are responsible for ligand binding and selectivity (see text), and

other motifs are important for signal transduction. The variable intracellular portions of the receptors allow them to interact with speciﬁc G

proteins.

VASOPRESSIN/OXYTOCIN RECEPTOR FAMILY 347

VP/OT peptides should interact with conserved

domains in the receptors, but perhaps things are not

this simple.

One amino acid appears to be crucial for peptide

agonist selectivity. This residue (marked with a V in

Figure 2) is found in the ﬁrst extracellular loop, and it

interacts with the eighth amino acid of the peptide

hormones (arginine and leucine in AVP and OT,

respectively). The aspartic acid or tyrosine residues

found at V in the human V2 and V1a/V1b receptors,

respectively, are responsible for their marked preferences

for AVP versus OT. The phenylalanine in this position in

the OT receptor accounts for its modest preference for

OT over AVP.

VP and OT antagonist-binding sites appear to be

different from the ones where the peptide hor-

mones bind.

FURTHER READING

Acher, R., and Chauvet, J. (1995). The neurohypophsial endocrine

regulatory cascade: Precursors, mediators, receptors, and effectors.

Front. Neuroendocrinol. 16, 237 –289.

Chini, B., and Fanelli, F. (2000). Molecular basis of ligand binding and

receptor activation in the oxytocin and vasopressin receptor family.

Exp. Physiol. 85S, 59S– 66S.

Hedges, B. S. (2002). The origin and evolution of model organisms.

Nature Rev. Genet. 3, 838–849.

Ivell, R., and Russell, J. A. (1995). Oxytocin: Cellular and Molecular

Approaches in Medicine and Research. Kluwer Academic/Plenum,

New York.

Zingg, H. H., Bourque, C. W., and Bichet, D. G. (1998). Vasopressin

and Oxytocin: Molecular Cellular, and Clinical Advances. Kluwer

Academic/Plenum, New York.

BIOGRAPHY

Michael J. Brownstein is Chief of the Laboratory of Genetics,

National Institute of Mental Health, NIH, Bethesda, MD.

He received his Ph.D. and M.D. degrees from the University

of Chicago and did postdoctoral training with Julius Axelrod.

His research has been in the areas of neurobiology, endocrinology,

genetics, and genomics. He and his co-workers are known for their

studies of vasopressin and oxytocin biosynthesis, and for cloning the

vasopressin and oxytocin receptors.

348 VASOPRESSIN/OXYTOCIN RECEPTOR FAMILY

V-ATPases

Michael Forgac

Tufts University School of Medicine, Boston, Massachusetts, USA

The V-ATPases (or vacuolar (H

)-ATPases) are ATP-driven

proton pumps whose primary function is to acidify intracellu-

lar compartments in eukaryotic cells, although they have

also been identiﬁed in the plasma membrane of certain cells.

V-ATPases have been shown to play a crucial role in a variety

of normal cellular processes as well as a number of human

diseases. The structure, mechanism, and regulation of these

proton pumps have been the topics of intense study.

V-ATPase Function

FUNCTION OF INTRACELLULAR

V-ATPASES

V-ATPases have been identiﬁed in many intracellular

compartments, including endosomes, lysosomes, Golgi-

derived vesicles and secretory vesicles. V-ATPases within

endosomal compartments are important for the process

of receptor-mediated endocytosis (Figure 1). During

receptor-mediated endocytosis, cells take up ligands

(such as the cholesterol carrying complex low density

lipoprotein, or LDL) from their environment by binding

them to receptors on the cell surface and clustering these

receptors in specialized regions of the plasma membrane

which then invaginate into the cell. Following this

internalization, the ligand-receptor complexes are

exposed to a low pH within the endosome that causes

the internalized ligand to dissociate from its receptor.

This dissociation allows the receptor to recycle to the

plasma membrane (where it is reutilized) and the

ligand to proceed to the lysosome, where it is degraded.

The low pH within the endosome is generated by the

V-ATPase.

Acidiﬁcation of endosomes is also important in the

formation of carrier vesicles that carry the released

ligands from early to late endosomal compartments, and

in the delivery of newly synthesized lysosomal enzymes

from the Golgi to lysosomes. The latter process involves

the binding of these enzymes to mannose-6-phosphate

receptors in the trans-Golgi followed by their delivery to

an endosomal compartment. Within this compartment,

the low pH created by the V-ATPases causes dissociation

of the lysosomal enzymes from their receptors, allowing

delivery of the enzymes to the lysosome and recycling of

the receptors to the trans-Golgi. Finally, endosomal

acidiﬁcation is involved in the entry of certain envelope

viruses (such as inﬂuenza virus) into cells. These viruses

bind to the surface of cells and are internalized by the

process of endocytosis. Upon exposure to a low pH, the

virus coat fuses with the endosomal membrane, releas-

ing the viral nucleic acid into the cytoplasm of the host

cell. Endosomal acidiﬁcation is therefore essential in the

process by which these viruses infect cells.

Lysosomes are the major compartment in which

degradation of proteins and other macromolecules

occurs in cells. The lysosomal enzymes responsible

for this degradation all require an acidic environment

to be active. This acidic environment is created by the

V-ATPases. Secretory vesicles, such as synaptic vesicles,

are also acidic compartments. Synaptic vesicles are

located at the synaptic terminal of nerve cells and release

neurotransmitters (that chemically trigger the next nerve

cell) by fusion with the plasma membrane. Neurotrans-

mitters become concentrated within synaptic vesicles by

transport proteins within the synaptic vesicle membrane

that utilize either the proton gradient or the membrane

potential generated by the V-ATPases to drive uptake of

the transmitter.

FUNCTION OF PLASMA

MEMBRANE V-ATPASES

Plasma membrane V-ATPases play an important role in

a number of normal and disease processes. In alpha-

intercalated cells in the kidney, V-ATPases are located in

the apical membrane where they pump protons into the

urine, thus helping to control the pH of the blood. A

genetic defect in this pump leads to a disease called renal

tubule acidosis, in which the kidney is unable to secrete

sufﬁcient acid. V-ATPases are also present in the plasma

membrane of osteoclasts, which are cells that function in

degradation of bone. These cells are essential during

development to facilitate bone remodeling. Plasma

membrane V-ATPases in osteoclasts create an acidic

extracellular environment that is necessary for bone

degradation to occur. A genetic defect in the V-ATPase in

osteoclasts leads to the human disease autosomal

recessive osteopetrosis, in which the inability to degrade

bone leads to severe skeletal defects and death.

Plasma membrane V-ATPases in macrophages and

neutrophils have been shown to help maintain a neutral

internal pH under conditions of severe acid load. In the

vas deferens, V-ATPases create a low pH environment

necessary for sperm development. V-ATPases in the

plasma membrane of tumor cells have also been

proposed to function in tumor invasion by providing

an acidic extracellular environment necessary for

secreted lysosomal enzymes to degrade extracellular

matrix. Finally, V-ATPases in intestinal cells in insects

create a membrane potential across the apical membrane

that is used to drive potassium transport into the gut.

V-ATPase Structure

The V-ATPase is a large complex composed of 13

different subunits. These subunits are arranged into two

separate domains termed V

and V

(Figure 2). The V

domain is made up entirely of subunits that are

peripheral to the membrane (i.e., not membrane-

embedded). This domain has a molecular mass of

, 640 kDa and contains eight different subunits (sub-

units A–H) of molecular mass 70– 13 kDa (Table I).

The V

domain is responsible for hydolysis of ATP,

which occurs on catalytic sites located on the three

copies of subunit A. There are therefore three catalytic

nucleotide binding sites per complex. The B subunits

(which are also present in three copies per complex) can

also bind nucleotides, but these sites are referred to as

“non-catalytic” sites, since they do not actually hydro-

lyze ATP. The function of these sites is not known,

but they may play a role in controlling the activity of the

V-ATPase. The A and B subunits are arranged in a

hexamer, like the segments of an orange, with alterna-

ting A and B subunits. ATP is hydrolyzed sequentially at

each of the three catalytic sites. The other subunits in the

domain (subunits C– H) function to connect the V

domain to the V

domain and are discussed here.

The V

domain is composed of ﬁve different subunits

(subunits a, d, c, c

,andc

)ofmolecularmass

100–17 kDa. All of the subunits in the V

domain

except subunit d are embedded in the membrane, and

thus require detergent for solubilization. The V

com-

plex has a molecular mass of 260 kDa and is responsible

for transport of protons across the membrane.

Receptor

Clathrin

coated

vesicle

Neuro-transmitter

Secretory

vesicle

Early

endosome

Recycling

vesicle

ATP

Late

endosome

Endosomal

carrier vesicle

Lysosome

M6P

receptor

Golgi

FIGURE 1 Function of intracellular V-ATPases. Acidiﬁcation of early endosomes by the V-ATPase facilitates receptor recycling following

endocytic uptake and formation of endosomal carrier vesicles. Recycling M6P receptors to the trans-Golgi is also low pH dependent. V-ATPase

activity is also required to drive neurotransmitter uptake and facilitates protein degradation in lysosomes.

350 V-ATPases

This proton transport only occurs when the V

domain

is attached to V

and is driven by the hydrolysis of ATP

in V

. Three of the subunits in the V

domain are called

proteolipid subunits (c, c

, and c

) because they are so

hydrophobic that they can be extracted from the

membrane using organic solvent mixtures, such as

chloroform:methanol. These subunits are almost com-

pletely embedded in the membrane and the polypeptide

chain of each one crosses the membrane four times

(these are called transmembrane segments). Buried in the

middle of one of the transmembrane segments of each of

the proteolipid subunits is a single essential glutamic

acid residue which is reversibly protonated and depro-

tonated during proton transport by the V-ATPases. Like

the A and B subunits in the V

domain, the proteolipid

subunits in the V

domain form a ring, with four copies

of subunit c and one copy each of subunit c

and c

. The

speciﬁc V-ATPase inhibitor baﬁlomycin has been shown

to bind to the proteolipid subunits of the V

domain.

In addition to the proteolipid subunits, the other V

subunit that is embedded in the membrane is the

100-kDa subunit a. This subunit is made up of two

domains. The carboxyl-terminal half of the molecule

contains nine transmembrane segments while the amino-

terminal half is a hydrophilic domain that is present on

the cytoplasmic side of the membrane. Like the proteo-

lipid subunits, the subunit a also contains amino acid

residues that are essential for proton transport. In par-

ticular, there is a positively charged arginine residue near

the middle of the seventh transmembrane segment of

subunit a that is absolutely required for proton transport

Lumen

MembraneV

domain

ATP

Cytoplasm

ADP + P

c''

FIGURE 2 Structure of the V-ATPases. The V-ATPases contain two domains. The V

domain is responsible for ATP hydrolysis and the V

domain

carries out proton transport across the membrane. Like the F-ATPases, the V-ATPases operate by a rotary mechanism in which ATP hydrolysis in

drives rotation of a central stalk which is connected to a ring of proteolipid subunits in V

. It is movement of the proteolipid ring relative to

subunit a that drives proton transport (see text).

TABLE I

Subunit Composition of the V-ATPase

Domain Subunit

Gene

(yeast)

(kDa)

Function/

location

A VMA1 69 Catalytic ATP binding site

B VMA2 58 Noncatalytic ATP

binding site

C VMA5 44 Peripheral stalk

D VMA8 29 Central stalk

E VMA4 26 Peripheral stalk

F VMA7 14 Central stalk

G VMA10 13 Peripheral stalk

H VMA13 54 Peripheral stalk

a VPH1/STV1 100 Proton translocation,

targeting

d VMA6 40 Cytoplasmic side

c VMA3 17 Proton translocation,

baﬁlomycin-binding site

VMA11 17 Proton translocation

VMA16 23 Proton translocation

V-ATPases 351

by the V-ATPases. The function of the remaining V

subunit d, which is tightly bound to the V

domain but is

not embedded in the membrane, is not known.

The V

and V

domains are connected by two stalks.

The central stalk is composed of the subunits D and F

whereas the peripheral stalk is composed of subunits C,

E, G, H, and the soluble domain of subunit a. The

function of these stalks is described below.

Mechanism of ATP-Driven Proton

Transport by the V-ATPases

The V-ATPases are believed to operate by a rotary

mechanism, similar to that demonstrated for the

F-ATPases (or ATP synthases), which are enzyme

complexes present in mitochondria, chloroplasts, and

bacteria that function in the reverse direction (that is in

proton-driven ATP synthesis). For the V-ATPases, ATP

hydrolysis in the V

domain drives rotation of the

central stalk (containing subunits D and F), which in

turn drives rotation of the ring of proteolipid subunits

relative to subunit a in the V

domain. Subunit a is held

ﬁxed relative to the A

hexamer of V

by the

peripheral stalk (or stator), composed of subunits C,

E, G, H, and the soluble domain of subunit a. It is

rotation of the ring of proteolipid subunits relative to

subunit a that drives active transport of protons from

the cytoplasmic to the lumenal side of the membrane. A

proton enters from the cytoplasmic side of the mem-

brane via a cytoplasmic access channel in subunit a and

protonates a buried carboxyl group on one of the

proteolipid subunits. ATP hydrolysis in V

forces

rotation of the proteolipid ring in the plane of the

membrane such that the protonated carboxyl group on

the proteolipid subunit reaches a second access channel

in subunit a that leads to the lumenal side of the

membrane. Interaction between this carboxyl group on

the proteolipid subunit and the buried arginine residue

of subunit a (which is positively charged) forces the

proton off of the proteolipid subunit into the lumenal

access channel, where it can be released to the lumenal

side of the membrane, thus completing the transport

cycle. In this way, the rotary motion driven by hydrolysis

of ATP is converted into unidirectional transport of

protons across the membrane.

Regulation of V-ATPase

Activity In Vivo

The activity of V-ATPases in different membranes in

the cell is known to be regulated such that the pH of

different intracellular compartments and the degree

of proton transport across the plasma membrane is

carefully controlled, but the mechanisms employed

in regulating V-ATPase activity in cells are still being

elucidated. One important mechanism of regulation

involves reversible dissociation of the V-ATPase com-

plex into its component V

and V

domains. In yeast,

dissociation occurs in response to removal of glucose

from the media, probably as a way to preserve

cellular energy stores. Dissociation has also been

demonstrated to occur in insects and in mammalian

cells. A second proposed regulatory mechanism

involves the formation of a disulﬁde bond between

two conserved cysteine residues located at the catalytic

site on the subunit A.

Differential targeting of V-ATPases to different

cellular membranes has also been proposed as a

means of controlling proton transport. This has been

shown to occur in intercalated cells in the kidney, where

exposure to a low pH causes the fusion of intracellular

vesicles containing the V-ATPase with the plasma

membrane, thus increasing proton transport out of

the cell into the renal ﬂuid. Differential targeting of

V-ATPases appears to be controlled by different iso-

forms of the subunit a. Thus the a3 isoform is able to

target the V-ATPase to the plasma membrane in

osteoclasts whereas the a4 isoform targets the V-ATPase

to the intercalated cell plasma membrane. It is

mutations in these isoforms that lead to the human

diseases osteopetrosis and renal tubule acidosis men-

tioned earlier. Isoforms have now been identiﬁed in

many of the V-ATPase subunits in mammalian cells,

and these have been shown to be expressed in both

tissue- and organelle-speciﬁc manner. This has led to

the expectation that speciﬁc inhibitors can be identiﬁed

that are selective in their ability to inhibit particular

V-ATPase complexes, which may in turn lead to cures

for diseases such as osteoporosis.