EVOLUTION OF GROWTH HORMONE, PROLACTIN AND THEIR RECEPTORS

Mike Wallis

Biochemistry and Biomedicine Group, School of Life Sciences, University of Sussex,

Brighton. U.K.

• Growth hormone (GH) and prolactin (PRL) are protein hormones from anterior pituitary

• GH and PRL show ~25% sequence identity and very similar 3D structure (4-helix bundle with up-up-down-down topology)

• Separate hormones in all vertebrates except cyclostomes; presumably arose by gene duplication

• GH promotes somatic growth; PRL stimulates lactation in mammals and has various actions in lower vertebrates

• Evolution in mammals shows (1) repeated duplications in some groups, and (2) variable (episodic) evolution rate

GH PRL

GROWTH HORMONE AND PROLACTIN

ORGANIZATION OF GH-LIKE GENES IN PRIMATES

Gene duplications gave varying families of GH-like genes in higher primates

PL = placental lactogen (= chorionic somatomammotropin, CS)

Human PL (85% sequence identity to hGH) expressed by placenta at high levels during pregnancy. GH-V (92% identity to hGH) expressed at modest levels during pregnancy.

GH gene clusters in NWM differ markedly from those in OWM/apes. Various factors, including phylogenetic analysis, indicate independent origins.

PRL locus contains a single gene in most mammals, including primates, but multiple duplications gave complex gene clusters in rodents and ruminants

PHYLOGENETIC TREE FOR MAMMALIAN GHS

Horse Elephant ManPig AlpacaOxSheep

0

0

17

01

12 2

Goat

0

Dog

0

76

Rat Mouse

41

5

2

0

7

Rabbit

7 3

Rhesus

2

0

Mole rat

0

25

50

75

100

Possum

5

11

0

Deer

0

32

Mill

ion

year

s be

fore

pre

sent

Chevrotain Dolphin MarmosetLoris

2

0

4

2

3

GP

12

GH evolution in mammals shows an episodic pattern with predominant near-stasis and occasional episodes of rapid change

Numbers of substitutions are shown on branches

PHYLOGENETIC TREES FOR MAMMALIAN GHs

rhesus monkey

marmoset

mouserathamster

mole ratground squirrel

guinea pigrabbitbushbaby

slow loris

human

whalehippopotamus

oxdeergiraffechevrotain

camelpighorsemink

dogcatpanda

bathedgehog

shrewarmadillo

elephanthyrax

possum

20 substitutions

ground squirrel

rhesus monkey

mouserathamster

mole rat

guinea pigrabbitbushbabyslow loris

humanmarmoset

whalehippopotamus

oxdeer

giraffechevrotain

camelpig

horsemink

dogcat

pandabat

hedgehogshrew

armadilloelephant

hyraxpossum

20 substitutions

Synonymous (dS) Nonsynonymous (dN)

For coding sequences bursts of rapid change for Nonsynonymous but not Synonymous substitutions

Trees constructed using codeml method of Yang. For thick branches nonsynonymous rate /synonymous rate (dN/dS; essentially rate of protein evolution relative to underlying rate) is significantly elevated

PROLACTIN EVOLUTION

HorseElephant ManPigCamelOxSheep

1

3

37

121 3

Goat

3

Cat

10

34

Rat Mouse

8 22

21

59

1

14

0

25

50

75

Rabbit

8 51 10

Macaque

0

3

Hamster

100

9

4

Mill

ion

year

s be

fore

pre

sent

Possum

Prolactin evolution is also episodic. Some bursts of rapid change

coincide with those seen for GH, but others are unique to prolactin

‘front’‘back’

Rc 1 Rc 2

GH

membrane

GH AND ITS RECEPTOR

3hhrDe Vos et al 1992

A homodimeric type 1 cytokine receptor

No evidence for duplication of GHR or PRLR in mammals, possibly because genes are large (~175 kb) compared with genes for GH and PRL (2-10 kb). Gene duplication giving ancestors of GHR and PRLR may have resulted from whole genome duplication early in vertebrate evolution

PHYLOGENETIC TREES FOR GH AND GHRBranch lengths from nonsynonymous substitutions (dN) from codeml; thick branches - dN/dS elevated significantly

PHYLOGENETIC TREES FOR PRL AND PRLRBranch lengths from nonsynonymous substitutions (dN) from codeml; thick branches - dN/dS elevated significantly

Bottom view (away from membrane)

top view(towards membrane)

front view

back view

ELEPHANT PROLACTIN/RECEPTOR COMPLEX

dN/dS* (overall) pPRL 0.61 (0.22) < 0.001PRLR 0.44 (0.36) > 0.05GH 0.039 (0.090) > 0.05GHR 0.30 (0.27) > 0.05

Statistical evaluation and dN/dS* ratios determined using codeml method

Residues changing on the lineage to elephant PRL shown in yellow

* Nonsynonymous substitution rate /synonymous substitution rate

Non-random distribution of substitutions

Based on structure 3npz: hPRL:rPRLR2

(van Agthoven et al 2010)

ARMADILLO GH/RECEPTOR COMPLEX

Non-random distribution of substitutions

front viewtop view

(towards membrane

back view

Bottom view (away from membrane)

dN/dS (overall) pPRL 0.31 (0.22) > 0.05PRLR 0.48 (0.36) > 0.05GH 0.48 (0.090) < 0.001GHR 0.38 (0.27) > 0.05

Based on structure 3hhr: hGH:hGHR2

(de Vos et al 1992)

BRANCH TO HIGHER PRIMATES GH/RECEPTOR COMPLEX

dN/dS (overall) pGH 0.43 (0.090) < 0.001GHR 0.97 (0.27) < 0.001 ecd 1.23 (0.24) < 0.001 icd 0.77 (0.27) < 0.01

GH:GHR

front view

back view

top view

bottom view

Substitutions in PRLR ecdGH:GHR

Based on structure 3hhr: hGH:hGHR2

(de Vos et al 1992)

Substantial proportion of substitutions in receptor binding sites

EVOLUTIONARY TREE FOR GHS AND PLS IN PRIMATESDuplications of the GH-gene were followed by episodes of rapid adaptive evolution

For ligands, many substitutions (subs) in binding sites (bs). Branch lengths based on dN values from codeml.Numbers on branches: amino acid substitutions (subs in bs)

EVOLUTIONARY TREE FOR GHS AND PLS IN PRIMATES

hGH acquires lactogenic activity

GH D171

GH T175

GHR W104

GHR D126

GHR R43L -> R

Branch to OWM/apes

Substitution 18Q->H (br to higher primates) allows Zn2+ coordination, required for binding to PRLR

hGHV loses lactogenic activitySubstitutions 18H -> R & 21H -> Y (branch to hGHV) prevent Zn2+ coordination, and binding to PRLR

hGH:hPRLR : 1bp3 Somers et al (1994)

EVOLUTIONARY TREE FOR GHS AND PLS IN PRIMATES

hPL loses somatogenic activity

GH D171

GH T175

GHR W104

GHR D126

GHR R43L -> R

Branch to OWM/apes

9 substitutions on branch to PLs/CSs, 6 of which are in binding sites. All potentially decrease binding by decreased hydrophobic interactions, loss of ion pairing or introduction of ionic repulsion

hGH:hGHR2 - 3hhr De Vos et al. 1992

CONCLUSIONS

• In mammals GH and PRL genes underwent multiple duplications on at least 4 occasions, giving complex gene clusters. Corresponding duplications of receptor genes are not seen.

• Evolution of GH and PRL shows prolonged periods of 'near stasis' and occasional episodes of rapid change. Evolution of their receptors also shows periods of rapid change, some of which correspond to those in the ligands, suggesting coevolution (e.g. human GH, ruminant PRL), others do not (e.g. armadillo GH, elephant PRL).

• GH gene duplications giving rise to placental lactogens etc occurred fairly late in primate evolution, independently in NWM and OWM/apes. Some substitutions occurring during the episodes of rapid evolution can be related to functional changes.

ACKNOWLEDGEMENTS

Sussex: Alex Lioupis, Zoe Maniou, Caryl Wallis

Monterrey, Mexico: Hugo A. Barrera-Saldaña, Irám Rodríguez-Sánchez, Antonio Pérez-Maya

EVOLUTIONARY TREE FOR GHS AND PLS IN PRIMATESThe basis for species specificity: GH 171 H->D on branch to higher primates does not affect binding to receptor; Subsequent GHR 43 L->R on branch to OWM/apes prevents binding of non-primate GH, but not human GH. (Souza et al. 1995)

GH D171

GH T175

GHR W104

GHR D126

GHR R43L -> R

Branch to OWM/apes

hGH:hGHR - 3hhrDe Vos et al. 1992

EVOLUTION OF PRIMATE GH GENE CLUSTERS

duplication and divergence

pseudogenization

duplication, gene conversion and pseudogenization

duplication and divergence

duplication and divergence

prosimian

intermediate 1

intermediate 2

orangutan

human

rhesus monkey

Two rounds of duplication and divergence were followed by divergent evolution of the clusters in orangutan, macaque and human

INDEPENDENT DUPLICATION OF GH GENE IN NEW-WORLD MONKEYS AND OLD-WORLD MONKEYS/APES

Phylogenetic analysis shows that GH-like genes in marmoset cluster together, with exclusion of all GH-like genes in OWM/apes

AMINO ACID SEQUENCES OF SOME MAMMALIAN GROWTH HORMONES

GH sequences are mostly strongly conserved, with some important exceptions

10 20 30 40 50 60 70 80 Pig FPAMPLSSLFANAVLRAQH LHQLAADTYKEFERAYIPEG QRYS-IQNAQAAFCFSETIP APTGKDEAQQRSDVELLRFSHorse ------------------- -------------------- ----•--------------- -------------M------ Dog ------------------- -------------------- ----•--------------- --------------------Mole rat -------N----------- -------------------- ----•--------------- -----E-------M------ Ox A----S--G----------- --------F-----T----- ----•---T-V--------- -----N----K--L----I- Sl loris ------------------- -------------------- ----•--------------- -------------M------ Man --TI---R--D--M---HR -----F---Q---E----KE -K--FL--P-TSL----S-- T-SNRE-T--K-NL----I-

90 100 110 120 130 140 150 160 Pig LLLIQSWLGPVQFLSRVFTN SLVFGTSD-RVYEKLKDLEE GIQALMRELEDGSPRAGQIL KQTYDKFDTNLRSDDALLKN Horse ------------L------- --------•------R---- -------------------- -------------------- Dog -------------------- --------•----------- -------------------- -------------------- Mole rat -------------------- --------•--F-------- -------------L----L- ----------M--------- Ox ----------L--------- --------•----------- --L---------T------- ----------M--------- Sl loris ------------L------- ---L----•----------- ---------------V---- -------------------- Man --------E-----RS--A- ---Y-A--SN--DL------ ---T--GR-------T---F ----S-----SHN-------

170 180 190 DIFFSPig YGLLSCFKKDLHKAETYLRV MKCRRFVESSCAFHorse -------------------- ------------- 3Dog -------------------- ------------- 0Mole Rat -------------------- ------------- 7 Ox -------R-----T------ ------G-A---- 19 Sl loris -------------------- ------------- 4 Man ----Y--R--MD-V--F--I VQ--•S--G--G- 62

PROLACTIN GENE CLUSTERS IN RODENTS AND RUMINANTS

GENE SIZES

GH 2kb 5 exons (chr 17)

PRL 10kb 5 exons (chr 6)

GHR 174kb 10 exons (chr 5)

PRLR 175kb 10 exons (chr 5)

Sizes and locations of genes in human

GH EVOLUTION IN PRIMATES

pig slow loris marmoset macaque manorangutanPLs PLs & GHV

3

4

0

0

12

76

00

4

2

Gene duplications

A burst of rapid change followed divergence of prosimians, but preceded divergence of new-world and old-world monkeys, and GH gene duplications.

55

Branch to ruminants prolactin/receptor complex

dN/dS (overall) pPRL 0.56 (0.22) < 0.001PRLR 1.13 (0.36) < 0.001 ecd 1.02 (0.29) < 0.001 icd 1.22 (0.37) < 0.001 GH 0.34 (0.090) < 0.001GHR 0.52 (0.27) < 0.01 ecd 0.86 (0.24) < 0.001 icd 0.27 (0.27) n.s.front view

back view

top view(towards membrane)

bottom view

Substitutions in PRLR ecd

PRL

Branch to higher primates GH/receptor complex

dN/dS (overall) pPRL 1.04 (0.22) < 0.001PRLR 0.75 (0.36) < 0.001 ecd 0.93 (0.29) < 0.001 icd 0.64 (0.37) > 0.05GH 0.43 (0.090) < 0.001GHR 0.97 (0.27) < 0.001 ecd 1.23 (0.24) < 0.001 icd 0.77 (0.27) < 0.01

GH

front view

back view

top view

bottom view

Substitutions in PRLR ecd

EVOLUTIONARY TREE FOR GHS AND PLS IN OWM/APES

slow loris GH

marmoset GH

macaque GH-N

human GH-N

orangutan GH-N

macaque GH V

human GH-V

orangutan GH-V

orangutan PL-B

human PL-A

human PL-B

macaque CS1

macaque CS2

macaque CS3

0.1

slow loris GH

marmoset GH

macaque GH-N

human GH-N

orangutan GH-N

macaque GH V

human GH-V

orangutan GH-V

orangutan PL-B

human PL-A

human PL-B

macaque CS1

macaque CS2

macaque CS3

synonymous nonsynonymous

0.1

Duplications of the GH-gene were followed by episodes of rapid adaptive evolution

PRBranch to higher primates PRL/receptor complexL-PRLR

dN/dS (overall) pPRL 1.04 (0.22) < 0.001PRLR 0.75 (0.36) < 0.001 ecd 0.93 (0.29) < 0.001 icd 0.64 (0.37) > 0.05GH 0.43 (0.090) < 0.001GHR 0.97 (0.27) < 0.001 ecd 1.23 (0.24) < 0.001 icd 0.77 (0.27) < 0.01

3D MODEL OF GH-Rc COMPLEX

human armadillo

Substitutions (yellow) are distributed in a non-random fashion. In human they are associated mainly with hormone-receptor interfaces, reflecting differences in specificity. In armadillo they occur mainly on the side away from the receptor and membrane, possibly reflecting interaction with another protein.

Based on structure of de Vos et al (1992)

FUNCTION SWITCHING - A MECHANISM FOR RAPID SEQUENCE EVOLUTION

X

X

XX

X

X

X

X

X

X

X

X

X

X

X

X

XX

Adaptation for 1 function Adaptation for 2 functions

Etc.

If GH acquired a second function, the importance of which fluctuated over time, each switch would lead to adaptation and additional substitutions. Repeated fluctuations would lead to substantial sequence change with relatively little change in function.

EVOLUTIONARY TREE FOR PROLACTIN IN PRIMATES

0.1 substitution

possum

dog

lemur

galago

slow loris

tarsier

marmoset

baboon

macaquegibbon

orangutan

gorilla

man

chimp

0.1 substitution

Synonymous Nonsynonymous

possum

dog

lemur

galago

slow loris

tarsier

marmoset

baboon

macaque

gibbon

orangutan

gorilla

manchimp

In primates prolactin shows a modest episode of rapid evolution but, unlike GH, no gene duplications

ACKNOWLEDGEMENTS

Sussex:Alex LioupisZoe ManiouCaryl Wallis

Monterrey, Mexico:Hugo A. Barrera-SaldañaIrám Rodríguez-SánchezAntonio Pérez-Maya