The Groove between the α- and β-Subunits of Hormones with Lutropin (LH) Activity Appears to Contact the LH Receptor, and Its Conformation Is Changed during Hormone Binding

Abstract

Gonadotropins are heterodimeric glycoprotein hormones that control vertebrate fertility through their actions on gonadal lutropin (luteinizing hormone, LH) and follitropin (follicle-stimulating hormone, FSH) receptors. The beta-subunits of these hormones control receptor binding specificity; however, the region of the beta-subunit that contacts the receptor has not been identified. By a process of elimination we show this contact to be the portions of beta-subunit loops one and three found in a hormone groove created by the juxtaposition of the alpha- and beta-subunits. Most other regions of the beta-subunit can be recognized by antibodies that bind to human chorionic hormone (hCG)-receptor complexes or replaced without disrupting hormone function. Using a series of bovine LH/hCG and human FSH/hCG beta-subunit chimeras we identified key hCG beta-subunit residues in the epitopes of two antibodies that bind to hCG-receptor complexes. These epitopes include the surfaces of beta-subunit loops one and three near residue 74 on the outside of the hormone groove and parts of the C-terminal end of the "seat belt" that holds the two subunits together. The antibody that recognized residue 74 bound to receptor complexes containing most mammalian lutropins better than to the free hormones, an indication that the outside surface of the beta-subunit groove is altered during hormone binding. This region of the beta-subunit is furthest from the alpha-subunit and is recognized equally well in the free beta-subunit and in the heterodimer. Thus, the receptor associated increase in antibody binding appears due to an interaction of this portion of the beta-subunit with the receptor and not to an effect of the receptor on the relative positions of the alpha- and beta-subunits. Unlike most previous studies designed to identify portions of the beta-subunit likely to contact the LH receptor, this indirect approach provides data that are more easily interpreted because it does not rely on the use of mutations that disrupt hormone function. The approach described here should be valuable for studying the receptor interactions of other complex ligands.

Influence of Subunit Interactions on Lutropin Specificity

“…Characterization of the immunological properties of each ␣-subunit analog has been described (1). All but two of the ␤-subunit analogs have also been described (20,28). CLC89 -96, an hLH/hCG ␤-subunit chimera having hCG ␤-subunit residues 89 -92 replaced by their hLH counterparts, was made by swapping parts of the cDNA that encode hCG and CLC77-96 (28) at PvuII restriction sites common to both.…”

“…Antibodies were obtained from Dr. Canfield, Drs. Glenn Armstrong and Robert Wolfert (Hybritech Inc., CA), and Dr. Janet Roser (University of California at Davis, CA) as noted previously (1,20). The hLHR cDNA was obtained from Dr. Aaron Hsueh (Stanford University, CA) in a vector that was used without modification to make stable CHO cell lines that express the human receptor.…”

“…Based on the activities of chemically and enzymatically modified hormones (summarized by Pierce and Parsons (7)), synthetic hormone fragments (8 -12), and analogs prepared by site-directed mutagenesis (13)(14)(15)(16)(17)(18)(19), residues throughout both hormone subunits have been suggested to participate in essential high affinity hormone receptor contacts. Surprisingly, some hCG residues proposed to contact LH receptors are in regions recognized by monoclonal antibodies that bind to hCG-receptor complexes (1,20). Others thought to be essential for receptor contacts can be replaced without disrupting receptor binding.…”

Alternatively Folded Choriogonadotropin Analogs

“…3 (20,25). This surface of the ␤-subunit is distant from the seatbelt latch disulfide, an observation that explains why B111 and B112 bind hCG at the same time.…”

“…The ␤-subunit antibodies used to monitor these analogs recognize sites on loops 1 and/or 3, a portion of the native hCG ␤-subunit detected readily before and after it has combined with the ␣-subunit. The B111 epitope includes the seatbelt latch (20,25). Although this antibody bound each of the ␣-subunit analogs when they were combined with the native hCG ␤-subunit (not shown), it was unable to recognize hCG-␤C26A (Fig.…”