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 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

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)

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

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)

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

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

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 front view top view (towards membrane) 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 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)

ARMADILLO GH/RECEPTOR COMPLEX Non-random distribution of substitutions dN/dS (overall) p PRL 0.31 (0.22) > 0.05 PRLR 0.48 (0.36) > 0.05 GH 0.48 (0.090) < 0.001 GHR 0.38 (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

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 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

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 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

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

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

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

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

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 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- 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 DIFFS Pig YGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF Horse -------------------- ------------- 3 Dog -------------------- ------------- 0 Mole 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

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)

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

Branch to ruminants prolactin/receptor complex PRL dN/dS (overall) p PRL 0.56 (0.22) < 0.001 PRLR 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) < 0.001 GHR 0.52 (0.27) < 0.01 ecd 0.86 (0.24) < 0.001 icd 0.27 (0.27) n.s. top view (towards membrane) front view Substitutions in PRLR ecd back view bottom view

Branch to higher primates GH/receptor complex dN/dS (overall) p PRL 1.04 (0.22) < 0.001 PRLR 0.75 (0.36) < 0.001 ecd 0.93 (0.29) < 0.001 icd 0.64 (0.37) > 0.05 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 GH front view top view 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 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

PRBranch to higher primates PRL/receptor complex L-PRLR dN/dS (overall) p PRL 1.04 (0.22) < 0.001 PRLR 0.75 (0.36) < 0.001 ecd 0.93 (0.29) < 0.001 icd 0.64 (0.37) > 0.05 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

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 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.

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

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