Alternatively Folded Choriogonadotropin Analogs

Abstract

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...

Glycoprotein Hormone Assembly in the Endoplasmic Reticulum

“…Unlike the hCG heterodimer, which dissociated completely at pH 2 within 30 min at 37°C, only about 20% of the hCG/teFSH chimera heterodimer dissociated under these conditions. It was not as stable as heterodimers that contain an intersubunit disulfide, which do not dissociate under these conditions (13). These observations showed that we could distinguish heterodimers in which the seatbelt is latched to the ␣-subunit, heterodimers in which the seatbelt is latched to ␤Cys 5 , and heterodimers in which the seatbelt is latched to ␤Cys 26 by measuring differences in their stabilities at pH 2, 37°C.…”

“…Previously, we had found that the hCG seatbelt in hCG␤-C26A is latched readily by a wraparound mechanism to either of these ␣-subunit analogs when it is prevented from latching with ␤Cys 26 (13). By analogy, we reasoned that if heterodimers containing the salmon seatbelt strap were latched by a wraparound mechanism, s/hCG␤-St,C26A would be latched readily to both ␣-S43C and ␣-S92C.…”

“…Furthermore, the position of this part of the seatbelt depends primarily on the location of the seatbelt latch site, which is found in loop ␤1 of most vertebrate hormones and in the ␤-subunit NH 2 terminus in salmon FSH (1). This can be varied experimentally in hCG analogs by moving the seatbelt latch site to different parts of the ␣-subunit (13). Because this portion of the hCG seatbelt appears to contribute little to lutropin activity other than to keep the heterodimer intact, we refer to it as the "strap."…”

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

“…We have observed repeatedly that disulfide cross-linked hCG analogs are stable at pH 2, 37°C, for 30 min or longer; hCG and heterodimers that lack a disulfide cross-link under these conditions dissociate and become undetectable in heterodimerspecific assays (9,18). Therefore, before using cross-linked samples in binding or signal transduction assays, we routinely treated them at acid pH to destroy any heterodimers that lack a cross-link.…”

“…Materials secreted into the culture media were assayed by sandwich immunoassays (17) employing ␣-subunit antibody A113 for capture and radioiodinated ␤-subunit antibody B110 for detection. They were treated at acid pH to cause the dissociation of heterodimers that lack an intersubunit disulfide cross-link (18). Chinese hamster cells that overexpress the rat LHR were used to monitor the influence of the analogs on the ability of 125 I-hCG to bind LHR and to elicit cyclic AMP accumulation as reported previously (5,13,15).…”