Influence of Subunit Interactions on Lutropin Specificity

The luteinizing hormone/choriogonadotropin receptor, a seven-transmembrane receptor, is composed of two equal halves, the N-terminal extracellular exodomain and the C-terminal membrane-associated endodomain. Unlike most seven-transmembrane receptors, the exodomain alone is responsible for high affinity hormone binding, whereas signal is generated in the endodomain. These physical separations of hormonebinding and receptor activation sites are attributed to unique mechanisms for hormone binding and receptor activation of this receptor and its subfamily members. However, the precise hormone contact sites in the exodomain are unclear. In the preceding article (Hong, S., Phang, T., Ji, I., and Ji, T. H. (1998) J. Biol. Chem. 273, 13835-13840), a region immediately downstream of the N terminus of the exodomain was shown to be crucial for hormone binding. To test if the region interacts with the hormone, human choriogonadotropin (hCG) was photoaffinity-labeled with a peptide mimic corresponding to Gly 18 -Tyr 36 of the receptor. This peptide mimic specifically photoaffinity-labeled both the ␣-and ␤-subunits of hCG. Interestingly, hCG␣ was preferentially labeled. On the other hand, denatured hCG was not labeled, and a mutant analog of the peptide failed to label hCG. Furthermore, the affinity labeling was UVdependent and saturable, indicating the specificity of the photoaffinity labeling. Our results indicate that the region of the exodomain interacts with hCG and that the contact points are near both subunits of hCG. Particularly, the alternate residues (Leu 20 , Cys 22 , and Gly 24 ) are crucial for hCG binding. In addition, the results underscore the fact that there is a crucial hormone contact site outside of the popularly believed primary hormonebinding site that is composed of Leu-rich repeats and is located in the middle of the exodomain. Our observations are crucial for understanding the molecular mechanism through which the initial high affinity hormone binding leads to receptor activation in the endodomain.The LH 1 /CG receptor belongs to a subfamily of glycoprotein hormone receptors within the seven-transmembrane receptor family. Unlike most seven-transmembrane receptors, it is composed of two equal halves, the 341-amino acid-long extracellular N-terminal exodomain and the 334-amino acid-long membrane-associated C-terminal endodomain, which includes seven transmembrane helices (1, 2). The exodomain binds the hormones with high affinity (3-7) without hormone action (5, 8). The exodomain-hCG complex is thought to make a secondary contact with the endodomain, thus generating a signal (9). Therefore, the high affinity interaction of the exodomain and hCG is the crucial first step leading to signal generation and hormone action. However, only limited information is available regarding the precise hormone contact residues and sites in the exodomain. Three peptide mimics of the exodomain, peptide-(21-38), peptide-(102-115), and peptide-(253-266), attenuated 125 I-hCG binding to membranes expressing the LH/CG recept...

mentioning

confidence: 66%

Section: Resultsmentioning

confidence: 99%

The Amino-terminal Region of the Luteinizing Hormone/Choriogonadotropin Receptor Contacts Both Subunits of Human Choriogonadotropin

Phang

Kundu

Hong

et al. 1998

“…Alternative approaches, such as assembly of pZPB-pZPC complexes using either native pig zona proteins pretreated with glycosidases or recombinant pig zona proteins with foreign N-and O-glycans or ablated glycosylation sequons, are therefore required. By analogy to the gonadotropins (51), data from such experiments must be interpreted with caution as molecular modifications that alter the interaction between pZPB and pZPC subunits may in theory also diminish the avidity of reconstituted pZPBpZPC complexes for cognate zona-binding proteins.…”

Section: Discussionmentioning

confidence: 99%

Hetero-oligomerization-dependent Binding of Pig Oocyte Zona Pellucida Glycoproteins ZPB and ZPC to Boar Sperm Membrane Vesicles

Yurewicz¹,

Sacco²,

Gupta³

et al. 1998

136

The zona pellucida surrounding the pig oocyte contains two M r 55,000 glycoproteins, pZPB and pZPC, which are orthologues of mouse zona proteins ZP1 and ZP3, respectively. We previously reported that isolated boar sperm membrane vesicles possess high affinity binding sites for partially purified pZPB, but not pZPC. Interestingly, co-incubation experiments also implicated pZPB-pZPC complexes as potential ligands. We now report that when depleted of a minor pZPC contaminant by size exclusion chromatography, pZPB lacks independent binding activity. In solid phase binding assays employing immobilized boar sperm membranes, pZPB failed to compete with biotin-(pZPB؉pZPC) probe, and biotin-labeled pZPB yielded negligible binding. However, when co-incubated with pZPC prior to the binding assays, pZPB acted as a potent competitor, and biotin-labeled pZPB exhibited high affinity, saturable binding. Binding activity was attributed to pZPB-pZPC heterocomplexes, which were detected in co-incubation mixtures by size exclusion chromatography and Western blot analysis. In the pig, therefore, sperm membranes possess a zona-binding protein with high affinity sites for pZPB-pZPC heterocomplexes, but not free glycoprotein subunits. Consequently, associative interactions between zona molecules can contribute toward both the assembly of the zona matrix and generation of ligands important for sperm-zona interactions.The zona pellucida is a morphologically discrete, extracellular matrix which envelops and protects the oocyte and preimplantation embryo. This egg investment also actively participates in the fertilization process. The zona pellucida provides docking sites for species-specific sperm attachment and induces bound sperm to undergo the acrosome reaction, the preparatory event for zona penetration and eventual sperm-egg fusion. This intimate association of male and female gametes is mediated in part by sperm-adhesive glycoproteins within the zona matrix and complimentary zona-binding proteins on the sperm surface. Interestingly, although multiple gamete adhesion molecules have been characterized at the molecular level (1-11), the relative physiological importance of many remains a topic of considerable debate (12).Zonae pellucidae contain a repertoire of glycoproteins encoded by three gene families that are conserved across species. Adopting the terminology of Harris et al. (13), the three cDNA-predicted precursor polypeptides in the pig (10,13,14) are herein designated pZPA (79 kDa), pZPB (59 kDa), and pZPC (46 kDa). Due to post-translational processing, the three mature glycoproteins from isolated zonae appear on nonreducing SDS gels as two diffuse bands (15); pZPA runs at M r 90,000, whereas pZPB and pZPC co-migrate at M r 55,000. Electrophoretic or chromatographic protocols permit isolation of copurified pZPB and pZPC glycoproteins (15-17) and such pZP(BϩC) preparations exhibit several important biological activities, including: stimulation of the acrosome reaction in capacitated boar sperm (18), inhibition of sperm-zona att...

“…The first and eighth residues are always cysteine, the fifth and sixth residues are usually serine or threonine, and the seventh residue is always aspartic acid. Of the three variable residues that corresponding to hCG ␤Arg-95 appears to have the greatest influence on LHR interactions, we have found that the leucine at this position in bovine LH accounts for much of its inability to interact with the human LHR (22). Furthermore, replacing ␤Arg-95 with a negatively charged residue has the largest impact on the lutropin activity of bifunctional chimera analogs (12).…”

Section: Discussionmentioning

confidence: 80%

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

Bernard

Lin

Cao

et al. 2004

Self Cite

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...