1. EVOLUTION OF GROWTH HORMONE, PROLACTIN AND THEIR RECEPTORS
Mike Wallis
Biochemistry and Biomedicine Group,
School of Life Sciences, University of Sussex,
Brighton. U.K.

2. GROWTH HORMONE AND PROLACTIN
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

3. 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.
PRL locus contains a single gene in most mammals, including primates, but multiple duplications gave complex gene clusters in rodents and ruminants
PL = placental lactogen (= chorionic somatomammotropin, CS)

4. PHYLOGENETIC TREE FOR MAMMALIAN GHS
GH evolution in mammals shows an episodic pattern with predominant near-stasis and occasional episodes of rapid change
Horse
Elephant
Man
Pig
Alpaca Ox
Sheep 17 1 12 2
Goat
Dog 76
Rat
Mouse 4 5 7
Rabbit 3
Rhesus
Mole rat 25 50 75
100
Possum 11
Deer
Million years before present
Chevrotain
Dolphin
Marmoset
Loris GP
Numbers of substitutions are shown on branches

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
rhesus monkey
marmoset
mouse
rat
hamster
mole rat
ground squirrel
guinea pig
rabbit
bushbaby
slow loris
human
whale
hippopotamus ox
deer
giraffe
chevrotain
camel
pig
horse
mink
dog
cat
panda
bat
hedgehog
shrew
armadillo
elephant
hyrax
possum
20 substitutions
Synonymous (dS)
Nonsynonymous (dN)

6. PROLACTIN EVOLUTION
Prolactin evolution is also episodic. Some bursts of rapid change coincide with those seen for GH, but others are unique to prolactin
Horse
Elephant
Man
Pig
Camel Ox
Sheep 1 3 37 2
Goat
Cat 10 34
Rat
Mouse 8 22 21 59 14 25 50 75
Rabbit 51
Macaque
Hamster
100 9 4
Million years before present
Possum

7. GH AND ITS RECEPTOR A homodimeric type 1 cytokine receptor GH Rc 1
membrane
‘front’
‘back’
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
3hhr
De Vos et al 1992

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

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

10. ELEPHANT PROLACTIN/RECEPTOR COMPLEX
Non-random distribution of substitutions
front view
top view
(towards membrane)
dN/dS* (overall) p
PRL (0.22) <
PRLR (0.36) > 0.05
GH (0.090) > 0.05
GHR (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
back view
Bottom view
(away from membrane)
* Nonsynonymous substitution rate /synonymous substitution rate
Based on structure 3npz: hPRL:rPRLR2
(van Agthoven et al 2010)

11. ARMADILLO GH/RECEPTOR COMPLEX
Non-random distribution of substitutions
dN/dS (overall) p
PRL (0.22) > 0.05
PRLR (0.36) > 0.05
GH (0.090) < 0.001
GHR (0.27) > 0.05
top view
(towards membrane
front view
Based on structure 3hhr: hGH:hGHR2
(de Vos et al 1992)
Bottom view (away from membrane)
back view

12. BRANCH TO HIGHER PRIMATES GH/RECEPTOR COMPLEX
Substantial proportion of substitutions in receptor binding sites
GH:GHR
dN/dS (overall) p
GH (0.090) < 0.001
GHR (0.27) < 0.001
ecd (0.24) < 0.001
icd (0.27) < 0.01
Based on structure 3hhr: hGH:hGHR2
(de Vos et al 1992)
front view
top view
Substitutions in PRLR ecd
GH:GHR
back view
bottom view

13. 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)

14. 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
H -> D
Branch to higher primates
GHR R43
GH T175
L -> R
Branch to OWM/apes
hGHV loses lactogenic activity
Substitutions 18H -> R & 21H -> Y (branch to hGHV) prevent Zn2+ coordination, and binding to PRLR

15. 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
GHR W104
GH D171
GHR D126
H -> D
Branch to higher primates
GHR R43
GH T175
L -> R
Branch to OWM/apes

16. CONCLUSIONS ACKNOWLEDGEMENTS
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

18. 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
H -> D
Branch to higher primates
GHR R43
GH T175
L -> R
Branch to OWM/apes
hGH:hGHR - 3hhr
De Vos et al. 1992

19. 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
duplication and divergence
pseudogenization
duplication, gene conversion and pseudogenization
prosimian
intermediate 1
intermediate 2
orangutan
human
rhesus monkey

20. 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

21. AMINO ACID SEQUENCES OF SOME MAMMALIAN GROWTH HORMONES
GH sequences are mostly strongly conserved, with some important exceptions
Pig FPAMPLSSLFANAVLRAQH LHQLAADTYKEFERAYIPEG QRYS-IQNAQAAFCFSETIP APTGKDEAQQRSDVELLRFS
Horse • 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-
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
DIFFS
Pig YGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF
Horse
Dog
Mole Rat
Ox R-----T G-A
Sl loris
Man Y--R--MD-V--F--I VQ--•S--G--G

22. PROLACTIN GENE CLUSTERS IN RODENTS AND RUMINANTS

23. Sizes and locations of genes in human
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)

24. 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
macaque
orangutan
man
PLs & GHV 3 4 12 76 2
Gene duplications 55

25. Branch to ruminants prolactin/receptor complex
PRL
dN/dS (overall) p
PRL (0.22) < 0.001
PRLR (0.36) < 0.001
ecd (0.29) < 0.001
icd (0.37) < 0.001
GH (0.090) < 0.001
GHR (0.27) < 0.01
ecd (0.24) < 0.001
icd (0.27) n.s.
top view
(towards membrane)
front view
Substitutions in PRLR ecd
back view
bottom view

26. Branch to higher primates GH/receptor complex
dN/dS (overall) p
PRL (0.22) < 0.001
PRLR (0.36) < 0.001
ecd (0.29) < 0.001
icd (0.37) > 0.05
GH (0.090) < 0.001
GHR (0.27) < 0.001
ecd (0.24) < 0.001
icd (0.27) < 0.01 GH
front view
top view
Substitutions in PRLR ecd
back view
bottom view

27. EVOLUTIONARY TREE FOR GHS AND PLS IN OWM/APES
Duplications of the GH-gene were followed by episodes of rapid adaptive evolution
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
synonymous
nonsynonymous

28. PRBranch to higher primates PRL/receptor complex L-PRLR
dN/dS (overall) p
PRL (0.22) < 0.001
PRLR (0.36) < 0.001
ecd (0.29) < 0.001
icd (0.37) > 0.05
GH (0.090) < 0.001
GHR (0.27) < 0.001
ecd (0.24) < 0.001
icd (0.27) < 0.01

29. 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)

30. 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. X
Adaptation for 1 function
Adaptation for 2 functions
Etc.

31. EVOLUTIONARY TREE FOR PROLACTIN IN PRIMATES
In primates prolactin shows a modest episode of rapid evolution but, unlike GH, no gene duplications
0.1 substitution
possum
dog
lemur
galago
slow loris
tarsier
marmoset
baboon
macaque
gibbon
orangutan
gorilla
man
chimp
Synonymous
Nonsynonymous

32. ACKNOWLEDGEMENTS
Sussex:
Alex Lioupis
Zoe Maniou
Caryl Wallis
Monterrey, Mexico:
Hugo A. Barrera-Saldaña
Irám Rodríguez-Sánchez
Antonio Pérez-Maya