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

Abstract

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...

Model of Glycoprotein Hormone Receptor Ligand Binding and Signaling

“…the "determinant loop") and the ␣-subunit COOH terminus, this requires that the ligand be oriented perpendicular to the LRD (5), an orientation that does not lend itself well to models of signal transduction. Furthermore, it does not explain the findings that the ␣-subunit COOH terminus can be cross-linked to most residues of the hCG small seatbelt loop by a disulfide without altering hormone activity (76).…”

“…Antibody binding data also revealed that the conformation of the tip of ␤-subunit 3 is altered on receptor binding (53). In revising our initial model, we made use of unpublished data (75,76) obtained with a technique developed to probe distances of specific residues in all three hormones and their receptors (data described in Fig. 10).…”

“…This orientation of the hormone in the receptor complex accounts for the finding that much of ␣-subunit loop 2 appears to be near the receptor interface although few of its residues participate in essential receptor contacts (75). Finally, the position of the hormone accounts for the finding that the COOH-terminal end of the ␣-subunit can be cross-linked by a disulfide to most portions of the small seatbelt loop without disrupting the biological activity of hCG (76). Indeed, relatively few residues in this loop, notably Arg 95 and Asp 99 , appear to contact the receptor interface.…”

Human choriogonadotrophin protein core and sugar branches heterogeneity: basic and clinical insights

“…There are at least two peptide regions located between cysteine residues b38 -57 and b93 -100 (Prasad et al, 2007) named receptor-determined loops, within the hCGb which confer receptor specificity. The surface able to activate the hCG receptor would include the majority of the hCGa and the Asp-99 residue contained in loops b93 -100 (Bernard et al, 2004). The CTP hCGb, not important for receptor binding or in vitro signal transduction, is critical for in vivo biological response (Chen and Puett, 1991).…”