Photoaffinity Labeling of the Lutropin Receptor with Synthetic Peptide for Carboxyl Terminus of the Human Choriogonadotropin γ Subunit

Abstract

Human choriogonadotropin (hCG) consists of an alpha subunit and a beta subunit. The existing evidence from various studies using truncation, substitution, synthetic hormone peptides, and hCG crystals suggests that the C-terminal region of the alpha subunit contacts the luteinizing hormone/choriogonoadotropin (LH/CG) receptor and is involved in receptor activation. Despite a deluge of the speculation and the important role of the alpha C-terminal region, direct evidence for its interaction with the receptor has been elusive. Because of the significant biological activity, it is imperative to prove the interaction of the alpha C-terminal region. For this purpose, decamer peptides corresponding to the alpha subunit sequence from His83 to Ser92 (alpha 83-92) were derivatized with the N-hydroxysuccinimide ester of 4-azidobenzoylglycine (ABG) and radioiodinated. The resulting ABG-125I-alpha 83-92 was capable of binding and activating the LH/CG receptor. Furthermore, UV-sensitive ABG-125I-alpha 83-92 exclusively photoaffinity-labeled an approximately of 86-kDa molecule. This labeled molecule was shown to be the LH/CG receptor by various methods including immunoprecipitation by anti-LH/CG receptor antiserum. In addition, evidence is presented that the amino group of alpha Lys91 of alpha 83-92 is in such close proximity to a carboxyl group of the receptor that this pair is cross-linked to form an amide, a zero length cross-link. This low affinity contact of alpha 83-92 and the receptor is sufficient for receptor activation and is crucial for the full understanding of the mechanistics of the receptor activation steps.

Structural Requirements for Mutational Lutropin/Choriogonadotropin Receptor Activation

“…First, the glycoprotein hormone binds to the extracellular domain, and distinct portions of the hormone act as agonist on the transmembrane receptor core. This mechanism is supported by studies showing that human choriogonadotropin (hCG) and peptides derived from the hCG ␣-chain can directly activate an LHR mutant that lacks the ectodomain (6,7). Second, the ectodomain of glycoprotein hormone receptors functions as an inverse agonist keeping the TMD region in an inactive conformation.…”

Biological Activities of Tethered Equine Chorionic Gonadotropin (eCG) and Its Deglycosylated Mutants

“…The CTP of the hCG α-subunit is important in receptor binding and signal transduction [17][18][19][20]. Previously, it has been shown that removal of the O-glycosylated region of CTP from both eLH β [21] and hCGβ [22][23][24][25] either has no effect or leads to enhancement of receptor-binding activity of these modified hormones.…”

Consequences of Single-Chain Translation on the Structures of Two Chorionic Gonadotropin Yoked Analogs in α-β and β-α Configurations

“…Indeed, several studies with single-chain hCG (22,23) and hFSH (35) have now ascribed the primary function of the cystine knot to intracellular behavior and not to productive in vitro bioactivity. First generation yoked hormones were constructed as N-␤-␣-C so as to utilize the CTP of ␤ as a linker and to retain the free C terminus of ␣, which is reported to be important in receptor binding and activation (10)(11)(12)(13). YhCG1 was able to bind to and activate LHR with properties similar to those of the native heterodimer.…”

“…It is evident that heterodimerization is a prerequisite for biological activity, and the individual subunits cannot activate LHR (8). The seat-belt loop is intimately involved in conferring specificity (9), and the C terminus of ␣ is important in high-affinity receptor binding and activation (10)(11)(12)(13). These critical regions are located on one face of the hormone (4,5), which carries a net positive surface charge that is complementary to the receptor's net negative surface charge reported in several homology models of the extracellular domain (14,15).…”