Bioengineering of Human Thyrotropin Superactive Analogs by Site-directed “Lysine-scanning” Mutagenesis (2000)

We have previously engineered the first superactive analogs of human thyrotropin (hTSH) by using a novel design strategy. In this study, we have applied homology comparisons focusing on the αL3 loop of the common α-subunit of human glycoprotein hormones. Seven highly variable amino acid residues were identified, and charge-scanning mutagenesis revealed three previously unrecognized modification permissive domains and four gain-of-function lysine substitutions. Such gain-of-function mutations were hormone- and receptor-specific and dependent on location and basic charge. Cooperativity of individual substitutions was established in double and triple lysine mutants. In combinations of the most potent αL3 loop analog with two previously characterized loop analogs, a higher degree of cooperativity for the αL3 loop analog compared with both the αL1 loop analog and the hTSH-βL3 loop analog was observed. We demonstrated that spatially distinct regions of the common α-subunit contribute differentially to the interaction of hTSH with its receptor and that combinations of two modified loops on the same and on opposite sides of the hTSH molecule display similar increases in in vitro biopotency. In addition, combination of all three superactive loops showed cooperativity in receptor binding and activation resulting in the most potent hTSH superactive analog described to date. We have previously engineered the first superactive analogs of human thyrotropin (hTSH) by using a novel design strategy. In this study, we have applied homology comparisons focusing on the αL3 loop of the common α-subunit of human glycoprotein hormones. Seven highly variable amino acid residues were identified, and charge-scanning mutagenesis revealed three previously unrecognized modification permissive domains and four gain-of-function lysine substitutions. Such gain-of-function mutations were hormone- and receptor-specific and dependent on location and basic charge. Cooperativity of individual substitutions was established in double and triple lysine mutants. In combinations of the most potent αL3 loop analog with two previously characterized loop analogs, a higher degree of cooperativity for the αL3 loop analog compared with both the αL1 loop analog and the hTSH-βL3 loop analog was observed. We demonstrated that spatially distinct regions of the common α-subunit contribute differentially to the interaction of hTSH with its receptor and that combinations of two modified loops on the same and on opposite sides of the hTSH molecule display similar increases in in vitro biopotency. In addition, combination of all three superactive loops showed cooperativity in receptor binding and activation resulting in the most potent hTSH superactive analog described to date. human thyrotropin bovine thyrotropin human chorionic gonadotropin human luteinizing hormone total thyroxine polymerase chain reaction Chinese hamster ovary Human thyrotropin (hTSH)1 is a member of a family of structurally related pituitary and placental human glycoprotein hormones that also includes the gonadotropins human chorionic gonadotropin (hCG), human-luteinizing hormone (hLH), and human follicle-stimulating hormone. Each hormone consists of a heterodimer of an α-subunit and a noncovalently linked β-subunit, and the α-β heterodimer formation is required for full biological activity. Whereas the primary structure of the α-subunit is common to all four glycoprotein hormones, the β-subunit is unique and responsible for hormone specificity (1Pierce J.G. Parsons T.F. Annu. Rev. Biochem. 1981; 50: 465-495Crossref PubMed Scopus (1899) Google Scholar). More recent studies have suggested a potential role for the individual free subunits in pregnancy and tumor growth (2Thotakura N.R. Blithe D.L. Glycobiology. 1995; 5: 3-10Crossref PubMed Scopus (88) Google Scholar, 3Bidart J.M. Baudin E. Troalen F. Bellet D. Schlumberger M. Ann. Endocrinol. (Paris). 1997; 58: 125-128PubMed Google Scholar, 4Blithe D.L. Richards R.G. Skarulis M.C. Endocrinology. 1991; 129: 2257-2259Crossref PubMed Scopus (89) Google Scholar). The recent elucidation of the crystal structure of chemically deglycosylated hCG (5Lapthorn A.J. Harris D.C. Littlejohn A. Lustbader J.W. Canfield R.E. Machin K.J. Morgan F.J. Isaacs N.W. Nature. 1994; 369: 455-461Crossref PubMed Scopus (833) Google Scholar, 6Wu H. Lustbader J.W. Liu Y. Canfield R.E. Hendrickson W.A. Structure. 1994; 2: 545-558Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar) has revealed that glycoprotein hormones share structural features with a large number of growth factors such as platelet-derived growth factor, neurotrophin (nerve growth factor), and transforming growth factor β (7Sun P.D. Davies D.R. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 269-291Crossref PubMed Google Scholar) and are therefore considered members of the superfamily of cystine knot growth factors. Within each glycoprotein subunit, a central cystine knot motif consisting of three disulfide bonds can be identified together with two peripheral β-hairpin loops (L1 and L3) on one side and one long loop (L2) on the other side. Despite a large diversity of membrane receptors within the superfamily of cystine knot growth factors, there are marked similarities in the functional role of the peripheral loop segments despite a limited (approximately 10–15%) amino acid sequence homology between the glycoprotein hormones and other members of the superfamily. Specifically, clusters of positively charged residues within the loops have recently been implicated as important receptor binding domains for members of the neurotrophin and the transforming growth factor β family (8Ryden M. Murray-Rust J. Glass D. Ilag L.L. Trupp M. Yancopoulos G.D. McDonald N.Q. Ibanez C.F. EMBO J. 1995; 14: 1979-1990Crossref PubMed Scopus (113) Google Scholar, 9Griffith D.L. Oppermann H. Rueger D.C. Sampath T.K. Tucker R.F. Carlson W.D. J. Mol. Biol. 1994; 244: 657-658Crossref PubMed Scopus (6) Google Scholar). Recent structure-function studies of human thyrotropin from our laboratory (for reviews see Refs. 10Szkudlinski M.W. Grossmann M. Weintraub B.D. Trends Endocrinol. Metab. 1996; 7: 277-286Abstract Full Text PDF PubMed Scopus (30) Google Scholar, 11Szkudlinski M.W. Grossmann M. Weintraub B.D. Curr. Opin. Endocrinol. Diabetes. 1997; 4: 354-363Crossref Scopus (7) Google Scholar, 12Grossmann M. Weintraub B.D. Szkudlinski M.W. Endocr. Rev. 1997; 18: 476-501Crossref PubMed Scopus (120) Google Scholar) and others have characterized multiple functionally important domains in both subunits (Fig.1). We distinguished “specificity determining” domains in the hTSHβ “seat belt” (hTSHβ-(88–105) (13Grossmann M. Szkudlinski M.W. Wong R. Dias J.A. Ji T.H. Weintraub B.D. J. Biol. Chem. 1997; 272: 15532-15540Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar)), “modification non-permissive” (α33–38 (14Grossmann M. Szkudlinski M.W. Dias J.A. Xia H. Wong R. Puett D. Weintraub B.D. Mol. Endocrinol. 1996; 10: 769-779Crossref PubMed Google Scholar), α-helix α40–46 (14Grossmann M. Szkudlinski M.W. Dias J.A. Xia H. Wong R. Puett D. Weintraub B.D. Mol. Endocrinol. 1996; 10: 769-779Crossref PubMed Google Scholar, 15Leinung M.C. Reed D.K. McCormick D.J. Ryan R.J. Morris J.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9707-9711Crossref PubMed Scopus (22) Google Scholar), glycosylation site α52 (16Grossmann M. Szkudlinski M.W. Tropea J.E. Bishop L.A. Thotakura N.R. Schofield P.R. Weintraub B.D. J. Biol. Chem. 1995; 270: 29378-29385Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 17Fares F.A. Gruener N. Kraiem Z. Endocrinology. 1996; 137: 555-560Crossref PubMed Scopus (26) Google Scholar), α-carboxyl terminus α88–92 (18Cheng K.-W. Glazer A.N. Pierce J.G. J. Biol. Chem. 1973; 248: 7930-7937Abstract Full Text PDF PubMed Google Scholar, 19Parsons T.F. Pierce J.G. J. Biol. Chem. 1979; 254: 6010-6015Abstract Full Text PDF PubMed Google Scholar, 20Grossmann M. Szkudlinski M.W. Zeng H. Kraiem Z. Ji I. Tropea J.E. Ji T.H. Weintraub B.D. Mol. Endocrinol. 1995; 9: 948-958Crossref PubMed Google Scholar)), and “modification permissive” domains in two peripheral β-hairpin loops (α11–20 (21Szkudlinski M.W. Teh N.G. Grossmann M. Tropea J.E. Weintraub B.D. Nat. Biotechnol. 1996; 14: 1257-1263Crossref PubMed Scopus (98) Google Scholar), hTSHβ-(58–69) (22Grossmann M. Leitolf H. Weintraub B.D. Szkudlinski M.W. Nat. Biotechnol. 1998; 16: 871-875Crossref PubMed Scopus (31) Google Scholar)). The mutational analysis of modification permissive domains defined as regions that tolerate the introduction of nonconservative amino acid changes into hTSH without compromising hormone synthesis (10Szkudlinski M.W. Grossmann M. Weintraub B.D. Trends Endocrinol. Metab. 1996; 7: 277-286Abstract Full Text PDF PubMed Scopus (30) Google Scholar) has been particularly useful in recent structure-function studies. Based on evolutionary considerations, amino acid structure comparisons between species and between different glycoprotein hormones, as as homology we were to a design for superactive analogs of human thyrotropin with increases in both receptor binding as as were the first to be described for glycoprotein hormone member of the cystine knot growth factor superfamily and by the increases in for other such as growth hormone J.A. J. Mol. Biol. PubMed Scopus Google Scholar) A. E. M. S. A. R. EMBO J. 1996; PubMed Scopus Google Scholar) using design In to the of in to an of hormone the analysis of gain-of-function mutations is to be with to location and the and therefore in glycoprotein structure-function studies. In the study, we have our design for superactive analogs of human thyrotropin to the as peripheral β-hairpin loop of the common α-subunit of human glycoprotein hormones the αL3 the that introduction of positively basic residues into all four peripheral β-hairpin loops to with the hTSH receptor resulting in an in hormone binding and in in vitro biopotency. The of the were as to modification permissive domains within the common αL3 loop of human glycoprotein to amino acid residues substitutions to basic residues to the of superactive hTSH to αL3 loop mutations to between individual residues within this and to between the αL3 loop and other peripheral β-hairpin were from and and the were by The human into of the was from H. Ji of The hTSH in the was previously engineered in our laboratory Y. Szkudlinski M.W. R. Weintraub B.D. Endocrinology. 1995; PubMed Google Scholar). Chinese hamster ovary the hTSH receptor and different receptor were by of In addition, has been established using a by G.D. Y. D. PubMed Scopus Google Scholar), in amino acid residues unique for the hTSH have been was to the role of receptor-specific in the in hTSH analog was a from J. of and were from of the human was by the of mutagenesis with two as described previously Google Scholar), using and by with the human in the as of multiple mutations within the αL3 previously in the with double amino acid substitutions were as for and been in the of our studies (21Szkudlinski M.W. Teh N.G. Grossmann M. Tropea J.E. Weintraub B.D. Nat. Biotechnol. 1996; 14: 1257-1263Crossref PubMed Scopus (98) Google Scholar). were to the and were to charged residues as and of the of the with and and of the resulting of this into the human in the with was of the αL1 loop analog with the αL3 loop analog was by of the αL3 loop analog with and of the into the αL1 loop analog in the with the were using a were using the and for multiple of and the for of large introduction of multiple mutations was by by the of of were in with bovine and and were in with in the for and multiple within the αL3 loop and in the for the analog and hTSH in the using a the were to In a we and of an were resulting in a total of was first by of this by was using resulting in a of was into were were for all studies as were each hTSH and hTSH analogs were with four different different and hTSH by a between a and a were In addition, we a hTSH using the hTSH β-subunit by the and and using hTSH by the as S. Weintraub D. R. Richards S. J.M. J.M. PubMed Scopus Google Scholar). The binding of hTSH was by to bovine from a membrane by of hTSH using a to the by the the human thyrotropin receptor and the thyrotropin receptor with of amino acid residues were in in with bovine and and were for with of hTSH in a modified was to with and bovine The of into the was by as described previously J.C. Endocrinology. PubMed Scopus Google Scholar). In was by a previously J. Szkudlinski M.W. J. Thotakura N.R. Weintraub B.D. 1995; 5: PubMed Scopus Google Scholar). were with with has been J. Szkudlinski M.W. J. Thotakura N.R. Weintraub B.D. 1995; 5: PubMed Scopus Google Scholar) to and thyrotropin for the by of total thyroxine this was as an of hTSH and hTSH analogs, was by and the were thyroxine were by acid sequence of the primary amino acid structure of the common αL3 loop of human glycoprotein hormones between amino acid residues and was to potential for mutagenesis The to be and from the β-subunit on an hTSH homology (21Szkudlinski M.W. Teh N.G. Grossmann M. Tropea J.E. Weintraub B.D. Nat. Biotechnol. 1996; 14: 1257-1263Crossref PubMed Scopus (98) Google Scholar) using of hCG (5Lapthorn A.J. Harris D.C. Littlejohn A. Lustbader J.W. Canfield R.E. Machin K.J. Morgan F.J. Isaacs N.W. Nature. 1994; 369: 455-461Crossref PubMed Scopus (833) Google Scholar, 6Wu H. Lustbader J.W. Liu Y. Canfield R.E. Hendrickson W.A. Structure. 1994; 2: 545-558Abstract Full Text Full Text PDF PubMed Scopus (454) Google acid sequence of the common αL3 loop of human glycoprotein hormones between design of and multiple lysine substitutions in a In to the in the common αL1 loop of human glycoprotein hormones (21Szkudlinski M.W. Teh N.G. Grossmann M. Tropea J.E. Weintraub B.D. Nat. Biotechnol. 1996; 14: 1257-1263Crossref PubMed Scopus (98) Google Scholar), the in the common αL3 loop clusters of positively charged amino acid We therefore distinguished residues with higher amino acid structure higher introduction of nonconservative amino acid we the as for to our that introduction of nonconservative basic residues analogs, we to the amino acid residues each of the with lysine in lysine substitutions as and In a four of mutations were to four four and one thyrotropin and human thyrotropin analogs were with four and Based on the most the the was of of for the of hTSH as compared with between and for with substitutions and between and for with multiple substitutions within the αL3 of for and triple lysine substitutions is in and of hTSH analogs with multiple substitutions in different loops are in of the was highly between all four and for of increases in in the studies. In the of as as our we have hTSH amino acid residues for binding of the in and we to be distinct and between the for this of of hTSH and hTSH analogs for and triple lysine substitutions in four different of hTSH for each in a of of hTSH and hTSH analogs for combinations of different loops in three different of hTSH for each in a The of hTSH for each The of hTSH for each of lysine substitutions and within the common αL3 loop hTSH analogs all of were and the introduction of nonconservative amino acid residues in a of the of the hormone in a of its biological regions as modification permissive of lysine substitutions hTSH superactive analogs, as showed a in the hTSH required for in the and of were between hTSH and hTSH The vitro was by a in the hTSH required for a of in a membrane The four showed increases in in the substitutions and in an in for the gain-of-function and the in in vitro lysine substitutions were to analogs with multiple the in the hTSH required for in the for the the double and the triple the same in the for the double and the triple In to the lysine double and triple lysine substitutions in the αL3 loop that between and of for the we increases in with two triple lysine substitutions as the of to and to in gain-of-function as in A. In addition, the triple compared with the with three lysine the triple was for and specificity introduction of nonconservative amino acid residues In to the of substitutions to basic residues within the αL3 loop as we also characterized the of substitutions to charged amino acid residues and as as one with three acid residues We a in as compared with the by the hormone for the substitutions and as as for the triple our of a basic of biopotency. a in to our and for acid and for in a in the the four superactive lysine substitutions in the αL3 loop in a a hTSH receptor G.D. Y. D. PubMed Scopus Google Scholar) in amino acid residues unique for the hTSH receptor and in the been we of superactive that the of the thyrotropin receptor to be in superactive of the four lysine substitutions within the αL3 of by the two most potent αL3 loop analogs with triple lysine substitutions also to a We one of the two previously characterized most potent triple lysine substitutions for and combination with two other superactive peripheral β-hairpin loop analogs that been in our laboratory We previously an αL1 loop analog with four lysine substitutions and (21Szkudlinski M.W. Teh N.G. Grossmann M. Tropea J.E. Weintraub B.D. Nat. Biotechnol. 1996; 14: 1257-1263Crossref PubMed Scopus (98) Google and an hTSH-βL3 loop analog with three lysine substitutions and (22Grossmann M. Leitolf H. Weintraub B.D. Szkudlinski M.W. Nat. Biotechnol. 1998; 16: 871-875Crossref PubMed Scopus (31) Google Scholar)). a of the in vitro for the and the analog with the We a higher degree of for the αL3 loop analog the αL1 loop analog with the hTSH-βL3 loop in the by and was therefore potent showed an in in vitro of combination with the hTSH-βL3 loop the in for the loop combination as as for the loop combination was and between and was also by the receptor binding studies In to the combination of the analog the analog with the analog in similar increases in in vitro for the two resulting combinations of superactive loops on the same on opposite sides of the hTSH molecule the αL3 loop analog was potent the hTSH-βL3 loop analog that a higher degree of be established for the αL3 loop analog the hTSH-βL3 loop analog with the αL1 loop we all three as characterized peripheral β-hairpin loops We an in in from to and analogs and a in binding in for combinations of superactive peripheral loops between and of the The of the analog to our previously most potent loop combination was in vitro and receptor binding the most potent hTSH superactive analog described to date. the in for hTSH the and the superactive were highly potent in and increases in in to in in studies were the of this the of hTSH analogs suggested in the for a of such analogs for in of in and In the study, we have mutations in the common αL3 loop of human glycoprotein hormones resulting in gain-of-function thyrotropin Whereas studies using have suggested a role of this loop in receptor binding J. R. A. Biochem. Biophys. PubMed Scopus (22) Google Scholar, Dias J.A. 5: Google Scholar, Dias J.A. Mol. Endocrinol. PubMed Scopus Google Scholar), mutagenesis studies have limited S. Mol. Endocrinol. 1996; PubMed Scopus Google Scholar, Mol. Endocrinol. 1997; PubMed Scopus (6) Google Scholar, Liu Dias J.A. 1998; PubMed Scopus Google Scholar). We have modified a recently design for glycoprotein hormone superactive analogs for mutational analysis of the common αL3 from our laboratory and others suggested a role of the peripheral β-hairpin loops of cystine knot growth factors in receptor binding (8Ryden M. Murray-Rust J. Glass D. Ilag L.L. Trupp M. Yancopoulos G.D. McDonald N.Q. Ibanez C.F. EMBO J. 1995; 14: 1979-1990Crossref PubMed Scopus (113) Google Scholar, 9Griffith D.L. Oppermann H. Rueger D.C. Sampath T.K. Tucker R.F. Carlson W.D. J. Mol. Biol. 1994; 244: 657-658Crossref PubMed Scopus (6) Google Scholar, M.W. Teh N.G. Grossmann M. Tropea J.E. Weintraub B.D. Nat. Biotechnol. 1996; 14: 1257-1263Crossref PubMed Scopus (98) Google Scholar). We have previously hTSH and hCG superactive analogs with increases in both receptor binding and on the introduction of positively charged lysine and residues into the common αL1 and the hTSH-βL3 loop (21Szkudlinski M.W. Teh N.G. Grossmann M. Tropea J.E. Weintraub B.D. Nat. Biotechnol. 1996; 14: 1257-1263Crossref PubMed Scopus (98) Google Scholar, M. Leitolf H. Weintraub B.D. Szkudlinski M.W. Nat. Biotechnol. 1998; 16: 871-875Crossref PubMed Scopus (31) Google Scholar). The amino acid residues as for mutagenesis in studies were from sequence comparisons between species different of hormone between different hormones in a Whereas this evolutionary to be and to of for was on the of clusters of positively charged lysine be in amino acid sequence comparisons within the common αL3 loop of human glycoprotein hormones. highly variable residues multiple evolutionary changes be We that three regions with particularly between species and tolerate the introduction of nonconservative amino acid and were therefore to a In to mutagenesis that been previously in functional studies J.A. 1995; PubMed Scopus Google Scholar, Dias J.A. Mol. Endocrinol. 1995; PubMed Scopus Google Scholar, J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), the be particularly useful for of glycoprotein hormones to three modification permissive and four of of the lysine substitutions in this to gain-of-function mutants. In studies we have the analogs with lysine substitutions in the αL3 loop with the hCG β-subunit We a in the of hTSH and hCG by lysine substitutions in the common αL3 loop of human glycoprotein hormones, in for the hTSH hCG In addition, was for the for the in the of was potent as mutations in the α-subunit are hTSH in a can be related to interaction the peripheral location of such mutations that an interaction is is to that a basic in the and an in the in the of the in the formation of a We have therefore and lysine substitutions in the αL3 loop for to the by the hormone in the of a hTSH receptor G.D. Y. D. PubMed Scopus Google Scholar) amino acid residues unique for the hTSH receptor and in the structure of the been superactive of the hTSH analogs with the receptor be with this receptor that the hTSH receptor is in the superactive of the αL3 loop superactive of the of superactive characterized for the receptor with different receptor receptor a in the analysis of interaction of glycoprotein hormones. first for regions of interaction with of receptor be to of the receptor as for a In a regions of can be to a analysis by defined receptor a limited number of amino acid residues have been a individual charged residues between the and the receptor can be that this the superactive be to interaction between hTSH and the hTSH receptor with the of superactive thyrotropin in our gain-of-function were highly as introduction of lysine residues and and in the of superactive were also and as a with three lysine residues and showed similar in and was potent a triple in was by combination of three basic residues within the αL3 in to our the most potent αL1 loop analog been engineered by introduction of four basic substitutions (21Szkudlinski M.W. Teh N.G. Grossmann M. Tropea J.E. Weintraub B.D. Nat. Biotechnol. 1996; 14: 1257-1263Crossref PubMed Scopus (98) Google Scholar). crystal of deglycosylated the homology of and studies that amino acid residues and in the are and in with the β-subunit (5Lapthorn A.J. Harris D.C. Littlejohn A. Lustbader J.W. Canfield R.E. Machin K.J. Morgan F.J. Isaacs N.W. Nature. 1994; 369: 455-461Crossref PubMed Scopus (833) Google Scholar, 6Wu H. Lustbader J.W. Liu Y. Canfield R.E. Hendrickson W.A. Structure. 1994; 2: 545-558Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, M.W. Teh N.G. Grossmann M. Tropea J.E. Weintraub B.D. Nat. Biotechnol. 1996; 14: 1257-1263Crossref PubMed Scopus (98) Google Scholar, Dias J.A. Mol. Endocrinol. PubMed Scopus Google Scholar, Dias J.A. Endocrinology. PubMed Scopus (30) Google Scholar). introduction of basic residues in such the and contribute to the formation of a between two α-subunit an on binding to its The that the introduction of charged acid acid residues in all one characterized in a in is our of a vitro hTSH analog by of between and The of hTSH demonstrated in a with an amino acid to acid be to other a in with the that the of in the on the that an amino acid to acid the same in a in the different of the amino acid side chain also a role in In addition, is in to the of the αL3 with the most location of all amino acid residues in this for introduction of nonconservative amino acid from this study, a modification of be as the in in the of our More studies in the be to The of in vitro of the αL3 loop by of as as binding studies were we the of on the in vitro of analogs with the αL3 loop we have previously compared the of in the of in vitro of superactive analogs with mutations in the αL1 and loop (22Grossmann M. Leitolf H. Weintraub B.D. Szkudlinski M.W. Nat. Biotechnol. 1998; 16: 871-875Crossref PubMed Scopus (31) Google Scholar). studies revealed in increases in in vitro different be demonstrated to be the increases between and superactive hormone. we have previously the of αL1 and analogs on of and hormone in human using (21Szkudlinski M.W. Teh N.G. Grossmann M. Tropea J.E. Weintraub B.D. Nat. Biotechnol. 1996; 14: 1257-1263Crossref PubMed Scopus (98) Google Scholar). in increases were in to the of the to our we basic substitutions within one peripheral β-hairpin loop were that between the and the receptor resulting from the introduction of lysine residues into the are also that are resulting from The combination of the three as characterized superactive peripheral β-hairpin loops of human thyrotropin to our studies to the analysis of between The higher degree of cooperativity between the αL3 the αL1 loop analog with the hTSH-βL3 loop analog and the similar increases in between and combinations that two modified superactive peripheral loops in higher cooperativity two spatially of both the and the hTSH receptor the In addition, the that the combination of the superactive and the hTSH-βL3 loop analogs was potent combination of two loops the that all three engineered loops are in receptor binding and The first for this as an of our evolutionary in the design of superactive analogs of human thyrotropin a in the design of glycoprotein hormone the of this highly the that is to the of interaction by introduction of positively charged amino acid substitutions into the resulting in a binding by the hormone to higher binding in interaction analogs of hTSH are into hTSH structure-function also have a of potential and human thyrotropin has been basic and E. F. Davies T.F. Weintraub B.D. N. J. 1997; PubMed Scopus Google Scholar, I. D.J. Davies T.F. M. J. J. Weintraub B.D. J. Endocrinol. Metab. 1994; PubMed Scopus Google Scholar) as for the of for design for superactive analogs of human thyrotropin evolutionary and the for a thyrotropin with in and therefore and The from studies are to other glycoprotein hormones, also be useful for other members of the cystine knot growth factor superfamily. We are to for with the We Leitolf for and all of this study, the and of the We also for and