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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 AND PROLACTIN
•
•
•
GH
PRL
•
•
Growth hormone (GH) and prolactin
(PRL) are protein hormones from
anterior pituitary
GH and PRL show ~25% sequence
identity and very similar 3D structure (4helix 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
ORGANIZATION OF GH-LIKE GENES IN PRIMATES
Gene duplications gave varying families of GH-like genes in higher primates
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.
PL = placental lactogen (= chorionic somatomammotropin, CS)
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
GH evolution in mammals shows an episodic pattern with predominant
near-stasis and occasional episodes of rapid change
Goat Sheep Ox Deer Chevrotain Dolphin Pig Alpaca Rabbit
0
0
Million years before present
0
0
2
12
0
0
0
2
7
3
3
2
7
3
12
17
1
2
4
0
4
5
76
50
11
2
0
0
2
75
Numbers of substitutions
are shown on branches
100
Rat Mouse Mole rat Possum
0
1
25
Horse Elephant Dog Loris Marmoset Man Rhesus GP
5
PHYLOGENETIC TREES FOR MAMMALIAN GHs
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
Synonymous (dS)
mouse
rat
hamster
mole rat
ground squirrel
guinea pig
rabbit
bushbaby
slow loris
rhesus monkey
human
marmoset
whale
hippopotamus
ox
deer
giraffe
chevrotain
camel
pig
horse
mink
dog
cat
panda
bat
hedgehog
shrew
armadillo
elephant
hyrax
possum
20 substitutions
mouse
rat
hamster
mole rat
ground squirrel
guinea pig
rabbit
bushbaby
slow loris
Nonsynonymous (dN)
whale
hippopotamus
ox
deer
giraffe
chevrotain
camel
pig
horse
mink
dog
cat
panda
bat
hedgehog
shrew
elephant
hyrax
possum
20 substitutions
rhesus monkey
human
marmoset
armadillo
PROLACTIN EVOLUTION
Prolactin evolution is also episodic. Some bursts of rapid change
coincide with those seen for GH, but others are unique to prolactin
0
Goat Sheep Ox Camel Pig
3
1
2
1
1
3
Million years before present
Rabbit Elephant Horse Cat Man Macaque Rat Mouse Hamster Possum
25
3
8
51 10
21
14
0
34
4
59
1
75
9
100
22
10
37
50
8
3
GH AND ITS RECEPTOR
A homodimeric type 1 cytokine receptor
GH
ecd
Rc 1
tmd
Rc 2
membrane
icd
jak2
‘back’
jak2
‘front’
3hhr
De Vos et al 1992
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 GHR
Branch lengths from nonsynonymous substitutions (dN) from codeml;
thick branches - dN/dS elevated significantly
PHYLOGENETIC TREES FOR PRL AND PRLR
Branch lengths from nonsynonymous substitutions (dN) from codeml;
thick branches - dN/dS elevated significantly
ELEPHANT PROLACTIN/RECEPTOR COMPLEX
Non-random distribution of substitutions
dN/dS* (overall) p
PRL
0.61 (0.22)
< 0.001
PRLR 0.44 (0.36)
> 0.05
GH
0.039 (0.090) > 0.05
GHR 0.30 (0.27)
> 0.05
front view
top view
(towards membrane)
Bottom view
(away from
membrane)
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
back view
Based on structure 3npz:
hPRL:rPRLR2
(van Agthoven et al 2010)
ARMADILLO GH/RECEPTOR COMPLEX
Non-random distribution of substitutions
dN/dS (overall)
PRL 0.31 (0.22)
PRLR 0.48 (0.36)
GH
0.48 (0.090)
GHR 0.38 (0.27)
front view
p
> 0.05
> 0.05
< 0.001
> 0.05
top view
(towards membrane
Based on structure 3hhr:
hGH:hGHR2
(de Vos et al 1992)
back view
Bottom view (away from
membrane)
BRANCH TO HIGHER PRIMATES GH/RECEPTOR COMPLEX
Substantial proportion of substitutions in receptor binding sites
GH:GHR
dN/dS (overall) p
GH 0.43 (0.090) < 0.001
GHR 0.97 (0.27) < 0.001
ecd 1.23 (0.24) < 0.001
icd 0.77 (0.27) < 0.01
front view
top view
Based on structure 3hhr:
hGH:hGHR2
(de Vos et al 1992)
Substitutions in PRLR ecd
GH:GHR
back view
bottom view
EVOLUTIONARY TREE FOR GHS AND PLS IN PRIMATES
Duplications 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
Substitution 18Q->H (br to higher primates) allows
Zn2+ coordination, required for binding to PRLR
hGH:hPRLR : 1bp3 Somers et al (1994)
GHR W104
GH D171
GHR D126
GHR R43
L -> R
Branch to
OWM/apes
GH T175
hGHV loses lactogenic activity
Substitutions 18H -> R & 21H -> Y (branch to
hGHV) prevent Zn2+ coordination, and binding to
EVOLUTIONARY TREE FOR GHS AND PLS IN PRIMATES
hPL loses somatogenic activity
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
GH Q46 (46Q->E)
GH R16 (16R->Q)
GH F1 (1F->V)
GH I179 (179 I->M)
GH I4 (4I->V)
GHR
W104
GH D171
GH N12 (12N->H)
GHR D126
receptor 2
GHR R43
receptor 1
L -> R
Branch to
OWM/apes
GH T175
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 PRIMATES
The 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)
GHR W104
GH D171
GHR D126
GHR R43
L -> R
Branch to
OWM/apes
GH T175
hGH:hGHR - 3hhr
De Vos et al. 1992
EVOLUTION OF PRIMATE GH GENE CLUSTERS
Two rounds of duplication and divergence were followed by divergent
evolution of the clusters in orangutan, macaque and human
prosimian
duplication and divergence
intermediate 1
duplication and divergence
intermediate 2
pseudogenization
orangutan
duplication, gene conversion and pseudogenization
human
duplication and divergence
rhesus monkey
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
Pig
Horse
Dog
Mole rat
Ox
Sl loris
Man
10
20
FPAMPLSSLFANAVLRAQH
-------------------------------------------N----------A----S--G------------------------------TI---R--D--M---HR
30
40
LHQLAADTYKEFERAYIPEG
-----------------------------------------------------------------F-----T----------------------------F---Q---E----KE
50
60
QRYS-IQNAQAAFCFSETIP
----•------------------•------------------•------------------•---T-V------------•---------------K--FL--P-TSL----S--
70
80
APTGKDEAQQRSDVELLRFS
-------------M-----------------------------E-------M----------N----K--L----I-------------M-----T-SNRE-T--K-NL----I-
Pig
Horse
Dog
Mole rat
Ox
Sl loris
Man
90
100
LLLIQSWLGPVQFLSRVFTN
------------L------------------------------------------------------L--------------------L--------------E-----RS--A-
110
120
SLVFGTSD-RVYEKLKDLEE
--------•------R-----------•------------------•--F---------------•-------------L----•-------------Y-A--SN--DL------
130
140
GIQALMRELEDGSPRAGQIL
---------------------------------------------------L----L--L---------T---------------------V------T--GR-------T---F
150
160
KQTYDKFDTNLRSDDALLKN
------------------------------------------------M------------------M-------------------------------S-----SHN-------
Pig
Horse
Dog
Mole Rat
Ox
Sl loris
Man
170
180
YGLLSCFKKDLHKAETYLRV
----------------------------------------------------------------R-----T----------------------------Y--R--MD-V--F--I
190
MKCRRFVESSCAF
------------------------------------------G-A---------------VQ--•S--G--G-
DIFFS
3
0
7
19
4
62
PROLACTIN GENE CLUSTERS IN RODENTS AND RUMINANTS
GENE SIZES
Sizes and locations of genes in human
GH 2kb 5 exons (chr 17)
PRL 10kb 5 exons (chr 6)
GHR 174kb 10 exons (chr 5)
PRLR 175kb 10 exons (chr 5)
GH EVOLUTION IN PRIMATES
A burst of rapid change followed divergence of prosimians, but preceded
divergence of new-world and old-world monkeys, and GH gene duplications.
pig
slow loris
PLs
marmoset
orangutan
macaque
0
12
man PLs & GHV
0
4
2
4
Gene duplications
3
0
76
55
0
Branch to ruminants prolactin/receptor complex
PRL
front view
top view
(towards membrane)
PRL
PRLR
ecd
icd
GH
GHR
ecd
icd
dN/dS (overall)
0.56 (0.22)
1.13 (0.36)
1.02 (0.29)
1.22 (0.37)
0.34 (0.090)
0.52 (0.27)
0.86 (0.24)
0.27 (0.27)
p
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.01
< 0.001
n.s.
Substitutions in PRLR ecd
back view
bottom view
Branch to higher primates GH/receptor complex
GH
front view
top view
dN/dS (overall)
PRL
1.04 (0.22)
PRLR 0.75 (0.36)
ecd 0.93 (0.29)
icd
0.64 (0.37)
GH
0.43 (0.090)
GHR 0.97 (0.27)
ecd 1.23 (0.24)
icd
0.77 (0.27)
p
< 0.001
< 0.001
< 0.001
> 0.05
< 0.001
< 0.001
< 0.001
< 0.01
Substitutions in PRLR ecd
back view
bottom view
EVOLUTIONARY TREE FOR GHS AND PLS IN OWM/APES
Duplications of the GH-gene were followed by episodes of rapid adaptive evolution
nonsynonymous
synonymous
slow loris GH
slow loris GH
marmoset GH
marmoset GH
macaque GH-N
macaque GH-N
human GH-N
human GH-N
orangutan GH-N
orangutan GH-N
macaque GH V
macaque GH V
human GH-V
human GH-V
orangutan GH-V
orangutan GH-V
orangutan PL-B
orangutan PL-B
human PL-A
human PL-A
human PL-B
human PL-B
macaque CS3
macaque CS3
macaque CS2
0.1
macaque CS1
macaque CS2
0.1
macaque CS1
PRBranch to higher primates PRL/receptor complex
L-PRLR
dN/dS (overall)
p
PRL
1.04 (0.22)
PRLR 0.75 (0.36)
ecd 0.93 (0.29)
icd
0.64 (0.37)
GH
0.43 (0.090)
GHR 0.97 (0.27)
ecd 1.23 (0.24)
icd
0.77 (0.27)
< 0.001
< 0.001
< 0.001
> 0.05
< 0.001
< 0.001
< 0.001
< 0.01
3D MODEL OF GH-Rc COMPLEX
armadillo
human
Substitutions (yellow)
are distributed in a nonrandom 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
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.
Adaptation for 1 function
Adaptation for 2 functions
X
X
X
X
X
X
X
X
X
X
X
X
Etc.
X
X
X
X
X
X
EVOLUTIONARY TREE FOR PROLACTIN IN PRIMATES
In primates prolactin shows a modest episode of rapid evolution
but, unlike GH, no gene duplications
Synonymous
Nonsynonymous
possum
possum
dog
dog
lemur
lemur
galago
slow loris
galago
slow loris
tarsier
tarsier
marmoset
baboon
marmoset
baboon
macaque
gibbon
macaque
gibbon
orangutan
orangutan
gorilla
gorilla
chimp
man
0.1 substitution
chimp
man
0.1 substitution
ACKNOWLEDGEMENTS
Sussex:
Alex Lioupis
Zoe Maniou
Caryl Wallis
Monterrey, Mexico:
Hugo A. Barrera-Saldaña
Irám Rodríguez-Sánchez
Antonio Pérez-Maya