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EVOLUTION OF GROWTH HORMONE, PROLACTIN AND THEIR RECEPTORS

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

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EVOLUTION OF GROWTH HORMONE, PROLACTIN AND THEIR RECEPTORS

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  1. EVOLUTION OF GROWTH HORMONE, PROLACTIN AND THEIR RECEPTORS MikeWallis 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. GP Marmoset Chevrotain Loris Dolphin Goat Sheep Ox Deer Rabbit Horse Elephant Dog Man Rat Mouse Mole rat Alpaca Rhesus Possum Pig 0 0 0 2 0 0 2 1 1 2 4 12 3 3 0 0 7 2 0 25 0 4 7 3 5 12 Million years before present 17 76 50 11 2 0 2 0 75 5 100 PHYLOGENETIC TREE FOR MAMMALIAN GHS 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

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

  6. Sheep Ox Camel Rabbit Elephant Horse Cat Man Macaque Rat Mouse Hamster Possum Pig Goat 0 3 1 2 8 1 22 1 3 3 21 8 51 10 10 3 25 37 14 0 34 50 4 Million years before present 59 1 75 9 100 PROLACTIN EVOLUTION Prolactin evolution is also episodic. Some bursts of rapid change coincide with those seen for GH, but others are unique to prolactin

  7. GH AND ITS RECEPTOR A homodimeric type 1 cytokine receptor GH Rc 2 Rc 1 membrane ‘back’ ‘front’ 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 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 back view Residues changing on the lineage to elephant PRL shown in yellow Bottom view (away from membrane) front view * Nonsynonymous substitution rate /synonymous substitution rate Based on structure 3npz: hPRL:rPRLR2 (van Agthoven et al 2010) top view (towards membrane)

  11. 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

  12. 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

  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 • 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

  17. 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

  18. 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

  19. 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

  20. 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

  21. PROLACTIN GENE CLUSTERS IN RODENTS AND RUMINANTS

  22. 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)

  23. 0 0 12 4 2 4 3 0 76 0 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. orangutan pig slow loris PLs marmoset macaque man PLs & GHV Gene duplications 55

  24. 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

  25. 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

  26. 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 macaque CS2 macaque CS1 macaque CS1 0.1 0.1 EVOLUTIONARY TREE FOR GHS AND PLS IN OWM/APES Duplications of the GH-gene were followed by episodes of rapid adaptive evolution

  27. 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

  28. 3D MODEL OF GH-Rc COMPLEX armadillo human 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)

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

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

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

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