Human chorionic gonadotropin with C-elongated alpha-subunit retains full receptor binding and partial agonist activity

Marichatou, H.; Martinat, Nadine; Guillou, Florian; Combarnous, Yves

doi:10.1530/eje.0.1420402

Cited by 4 publications

(3 citation statements)

References 20 publications

(17 reference statements)

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“…We first focused on this C-terminal part of the a-subunit as deletion and mutagenesis studies had previously shown that this region in the human a-subunit was important for binding and bioactivity of gonadotropins (11-13, 29 -31). Moreover, hCG with its a-subunit elongated by 24 residues beyond the terminal serine displayed full receptor binding and a sixfold reduction in LH bioactivity assessed on rat Leydig cells (32). However, this study shows that the mutations introduced in the C-terminal part of a-dk did not abolish FSH activity of dkLH or eLH, showing that these unique positions in the equid a-subunits do not favor interaction with the FSH receptor.…”

Section: Discussionmentioning

confidence: 61%

Identification of amino-acids in the alpha-subunit first and third loops that are crucial for the heterospecific follicle-stimulating hormone activity of equid luteinizing hormone/choriogonadotropin

Chopineau¹,

Martinat²,

Gibrat³

et al. 2004

European Journal of Endocrinology

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Identification of amino-acids in the a-subunit first and third loops that are crucial for the heterospecific follicle-stimulating hormone activity of equid luteinizing hormone/choriogonadotropin M Chopineau, N Martinat, J F Gibrat 1 , C Galet, F Lecompte, F Foulon-Gauze, C Pourchet, F Guillou and Y Combarnous AbstractObjective: To identify amino-acids in the a-subunit important for expression of heterospecific FSH activity of horse (e) LH/choriogonadotropin (CG) (eLH) and donkey (dk) LH/CG (dkLH) (FSH/LH ratio ten times higher for eLH than for dkLH); this FSH activity absolutely requires an equid (donkey or horse) a-subunit combined with an equid b-LH subunit. Design: Chimeric a-subunits possessing the first 63 amino-acids of the porcine (p) and the last 33 amino-acids of the donkey a-subunit (ap-dk) and the inverse (adk-p) were constructed. Porcinespecific amino-acids were introduced by mutagenesis in donkey a-subunit at positions 70, 85, 89, 93 and 96 (adk5xmut), 18 (adk K18E ) or 78 (adk I78A ). Methods: These different a-subunits were co-transfected in COS-7 cells with b-eLH, b-dkLH and b-eFSH. The LH and FSH bioactivities of the dimers were then assessed in two heterologous in vitro bioassays. Results: ap-dk or adk-p exhibited FSH activity when co-expressed with b-eLH but not with b-dkLH. adk K18E or adk I78A gave hybrids with no FSH activity and important LH activity when expressed with b-dkLH. adk I78A /beLH displayed an FSH/LH ratio as low as that of dkLH. However, mutation at 78 in a-dk had no effect on FSH bioactivity when co-expressed with b-eFSH. Conclusions: Amino-acids present in both the first two-thirds and the last third of the a-subunit of equid LHs are involved in their heterologous biospecificity. Ile a78 exerts as strong an influence on it as the b102-103 residues. By contrast, this residue plays no role in the FSH specificity of eFSH.

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Section: Discussionmentioning

confidence: 61%

Identification of amino-acids in the alpha-subunit first and third loops that are crucial for the heterospecific follicle-stimulating hormone activity of equid luteinizing hormone/choriogonadotropin

Chopineau¹,

Martinat²,

Gibrat³

et al. 2004

European Journal of Endocrinology

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“…Fusion of peptides to this portion of the ␣-subunit has been shown to have varying effects on hormone activity (20,21). Therefore, when we began these studies we were concerned that the presence of a crosslink between the ␣CT and the hCG ␤-subunit might destroy heterodimer activity by a mechanism that was unrelated to the ability of this portion of the hormone to contact the LHR.…”

Section: Resultsmentioning

confidence: 99%

Only a Portion of the Small Seatbelt Loop in Human Choriogonadotropin Appears Capable of Contacting the Lutropin Receptor

Bernard

Lin

Cao

et al. 2004

Journal of Biological Chemistry

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Twenty residues of the human choriogonadotropin (hCG) ␤-subunit that are wrapped around ␣-subunit loop 2 like a "seatbelt" stabilize the heterodimer and enable the hormone to distinguish lutropin (LHR), follitropin, and thyrotropin receptors. The N-terminal portion of the seatbelt contains a small disulfide-stabilized loop needed for heterodimer assembly and is thought to mediate hCG-LHR interactions. To test the latter notion, we compared the LHR binding and signal transduction activities of hCG analogs in which the ␣-subunit C terminus (␣CT) was cross-linked to residues in the small seatbelt loop. Analogs having an intersubunit disulfide between a cysteine in place of ␣CT residue ␣Ser-92 and cysteines substituted for loop residues ␤Arg-94, ␤Arg-95, or ␤Ser-96 had high activities in LHR binding and signaling assays despite the fact that both portions of the hormone are thought to be essential for hCG activity. Use of a larger probe blocked hormone activity when the ␣CT was crosslinked to cysteines in place of residues ␤Arg-95 and ␤Asp-99, but not to cysteines in place of residues ␤Arg-94, ␤Ser-96, or ␤Thr-97. This suggested that the side chains of residues ␤Arg-95 and ␤Asp-99, which face in the same outward direction from the heterodimer, are nearer than the others to the LHR interface. The finding that residue 95 can be cross-linked to small ␣CT probes without eliminating hormone activity indicates its side chain does not participate in essential LHR contacts. We suggest that contacts between the small seatbelt loop and the LHR, if any, involve its backbone atoms and possibly the side chain of residue ␤Asp-99.The crystal structure of hCG 1 revealed that 20 residues of its ␤-subunit surround loop ␣2 like a "seatbelt" (1, 2). The seatbelt has a key role in the stability of the heterodimer; elimination of the disulfide that "latches" its C-terminal end to ␤Cys-26 in the subunit core disrupts heterodimer formation (3). Thus, glycoprotein hormones differ from most other heterodimeric proteins, which are stabilized by hydrophobic contacts or intersubunit disulfides. Whereas the evolutionary advantages of this unusual structural arrangement remain unknown, it may have facilitated the co-evolution of ligand-receptor pairs by permitting point mutations to alter the conformation of the heterodimer and thereby modulate its biological activity (4 -6).The seatbelt is divided into two regions that differ in their influence on the activities of lutropins, follitropins, and thyrotropins. Its N-terminal half contains a small disulfide-stabilized loop that has an important role in heterodimer assembly (7-9). Because a portion of this loop participates in hydrogen bonds with ␣-subunit loop 2 (1, 2, 10), mutations that affected its conformation or the stability of these hydrogen bonds would be expected to alter the positions of the subunits in the heterodimer. The seatbelt loop contains positively charged residues in mammalian lutropins and negatively charged residues in mammalian follitropins and thyrotropins. This led to the sp...

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“…The activities of analogs in which the seatbelt is latched to residues at or near the carboxyl terminus, albeit low, offer additional insights into the role of this hormone region in receptor interactions. The very end of the ␣-subunit carboxyl terminus appears to be exposed in the lutropin receptor complex as shown by the finding that a few residues can be added to this site without disrupting hormone activity (37). This portion of the hormone may be near the receptor interface, however, as shown by the finding that the presence of hCG ␤-subunit residues 118 -145, a region that contains several glycosylated serine residues in hCG, reduced the activity of hCG ϳ50-fold (38).…”

Section: Figmentioning

confidence: 99%

Alternatively Folded Choriogonadotropin Analogs

Xing

Lin

Jiang

et al. 2001

Journal of Biological Chemistry

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Most heterodimeric proteins are stabilized by intersubunit contacts or disulfide bonds. In contrast, human chorionic gonadotropin (hCG) and other glycoprotein hormones are secured by a strand of their ␤-subunits that is wrapped around ␣-subunit loop 2 "like a seatbelt." During studies of hCG synthesis in COS-7 cells, we found that, when the seatbelt was prevented from forming the disulfide that normally "latches" it to the ␤-subunit, its carboxyl-terminal end can "scan" the surface of the heterodimer and become latched by a disulfide to cysteines substituted for residues in the ␣-subunit. Analogs in which the seatbelt was latched to residues 35, 37, 41-43, and 56 of ␣-subunit loop 2 had similar lutropin activities to those of hCG; that in which it was latched to residue 92 at the carboxyl terminus had 10 -20% the activity of hCG. Attachment of the seatbelt to ␣-subunit residues 45-51, 86, 88, 90, and 91 reduced lutropin activity substantially. These findings show that the heterodimer can form before the ␤-subunit has folded completely and support the notions that the carboxylterminal end of the seatbelt, portions of ␣-subunit loop 2, and the end of the ␣-subunit carboxyl terminus do not participate in lutropin receptor interactions. They suggest also that several different architectures could have been sampled without disrupting hormone activity as the glycoprotein hormones diverged from other cysteine knot proteins.The heterodimeric placental glycoprotein hormone hCG 1 binds LHR and stimulates ovarian steroid synthesis during early pregnancy (1). The structure of hCG is known (2, 3), but the structures of the LHR and the hormone receptor complex remain to be elucidated. Each hCG subunit is divided into three elongated loops by cystine knots (2, 3), and the heterodimer is stabilized by a part of the ␤-subunit termed the "seatbelt" (2) that is wrapped around ␣-subunit loop 2. The composition of the seatbelt determines the receptor binding specificity of hCG (4 -6), but it is not known if this portion of the hormone contacts the receptor. The LHR is a G protein-coupled receptor that contains a large extracellular domain that binds hCG with high affinity and specificity (7). Based on its leucine-rich repeat motif (7), the LHR extracellular domain is usually presumed to be horseshoe-shaped, similar to ribonuclease inhibitor (8).The surfaces of hCG most likely to contact the LHR remain debated. The carboxyl-terminal end of the ␣-subunit, which is adjacent to a portion of the seatbelt in the heterodimer (2, 3), has been found to influence the affinity of all the glycoprotein hormones for their receptors (1, 9) and was proposed to be a receptor contact more than 25 years ago (1). Based partially on this observation, Jiang et al. (10) suggested that the long axis of hCG docks with the concave surface of a horseshoe-shaped extracellular domain. In their view, the hormone is perpendicular to the extracellular domain of the receptor such that the ␣-subunit carboxyl-terminal end, parts of ␤-subunit loop 2, and the seatbelt...

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Human chorionic gonadotropin with C-elongated alpha-subunit retains full receptor binding and partial agonist activity

Cited by 4 publications

References 20 publications

Identification of amino-acids in the alpha-subunit first and third loops that are crucial for the heterospecific follicle-stimulating hormone activity of equid luteinizing hormone/choriogonadotropin

Identification of amino-acids in the alpha-subunit first and third loops that are crucial for the heterospecific follicle-stimulating hormone activity of equid luteinizing hormone/choriogonadotropin

Only a Portion of the Small Seatbelt Loop in Human Choriogonadotropin Appears Capable of Contacting the Lutropin Receptor

Alternatively Folded Choriogonadotropin Analogs

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