Win Lin

Models of glycoprotein hormone receptor interaction

The glycoprotein hormones regulate reproduction and development through their interactions with receptors in ovarian, testicular, and thyroid tissues. Efforts to design hormone agonists and antagonists useful for treating infertility and hyperthyroidism would benefit from a molecular understanding of hormone-receptor interaction. The structure of a complex containing FSH bound to a fragment of its receptor has been determined at 2.9 Å resolution, but this does not explain several observations made with cell-surface G protein receptors and may reflect the manner in which FSH binds a short alternate spliced receptor form. We discuss observations that must be explained by any model of the cell-surface G protein-coupled glycoprotein hormone receptors and suggest structures for these receptors that satisfy these requirements. Glycoprotein hormones appear to contact two distinct sites in the extracellular domains of their receptors, not just the leucine-rich repeat domain. These dual contacts contribute to ligand binding specificity and appear to be essential for signal transduction. As outlined in this minireview, differences in the manners in which these ligands contact their receptors explain why some ligands and ligand analogs interact with more than one class of receptor and why some receptors and receptor analogs bind more than one ligand. The unique manner in which these ligands appear to interact with their receptors may have facilitated hormone and receptor co-evolution during early vertebrate speciation.

Glycoprotein Hormone Assembly in the Endoplasmic Reticulum I. THE GLYCOSYLATED END OF HUMAN α-SUBUNIT LOOP 2 IS THREADED THROUGH A β-SUBUNIT HOLE

Glycoprotein hormone heterodimers are stabilized by their unusual structures in which a glycosylated loop of the α-subunit straddles a hole in the β-subunit. This hole is formed when a cysteine at the end of a β-subunit strand known as the “seatbelt” becomes “latched” by a disulfide to a cysteine in the β-subunit core. The heterodimer is stabilized in part by the difficulty of threading the glycosylated end of the α-subunit loop 2 through this hole, a phenomenon required for subunit dissociation. Subunit combination in vitro, which occurs by the reverse process, can be accelerated by removing the α-subunit oligosaccharide. In cells, heterodimer assembly was thought to occur primarily by a mechanism in which the seatbelt is wrapped around the α-subunit after the subunits dock. Here we show that this “wraparound” process can be used to assemble disulfide cross-linked human choriogonadotropin analogs that contain an additional α-subunit cysteine, but only if the normal β-subunit latch site has been removed. Normally, the seatbelt is latched before the subunits dock and assembly is completed when the glycosylated end of α-subunit loop 2 is threaded beneath the seatbelt. The unexpected finding that most assembly of human choriogonadotropin, human follitropin, and human thyrotropin heterodimers occurs in this fashion, indicates that threading may be an important phenomenon during protein folding and macromolecule assembly in the endoplasmic reticulum. We suggest that the unusual structures of the glycoprotein hormones makes them useful for identifying factors that influence this process in living cells.

Glycoprotein hormone assembly in the endoplasmic reticulum: IV. Probable mechanism of subunit docking and completion of assembly.

The unique structures of human choriogonadotropin (hCG) and related glycoprotein hormones make them well suited for studies of protein folding in the endoplasmic reticulum. hCG is stabilized by a strand of its β-subunit that has been likened to a “seatbelt” because it surrounds α-subunit loop 2 and its end is “latched” by an intrasubunit disulfide bond to the β-subunit core. As shown here, assembly begins when parts of the NH2 terminus, cysteine knot, and loops 1 and 3 of the α-subunit dock reversibly with parts of the NH2 terminus, cystine knot, and loop 2 of the hCG β-subunit. Whereas the seatbelt can contribute to the stability of the docked subunit complex, it interferes with docking and/or destabilizes the docked complex when it is unlatched. This explains why most hCG is assembled by threading the glycosylated end of α-subunit loop 2 beneath the latched seatbelt rather than by wrapping the unlatched seatbelt around this loop. hCG assembly appears to be limited by the need to disrupt the disulfide that stabilizes the small seatbelt loop prior to threading. We postulate that assembly depends on a “zipper-like” sequential formation of intersubunit and intrasubunit hydrogen bonds between backbone atoms of several residues in the β-subunit cystine knot, α-subunit loop 2, and the small seatbelt loop. The resulting intersubunit β-sheet enhances the stability of the seatbelt loop disulfide, which shortens the seatbelt and secures the heterodimer. Formation of this disulfide also explains the ability of the seatbelt loop to facilitate latching during assembly by the wraparound pathway.

Glycoprotein Hormone Assembly in the Endoplasmic Reticulum III. THE SEATBELT AND ITS LATCH SITE DETERMINE THE ASSEMBLY PATHWAY

Vertebrate glycoprotein hormone heterodimers are stabilized by a strand of their β-subunits known as the “seatbelt” that is wrapped around loop 2 of their α-subunits (α2). The cysteine that terminates the seatbelt is “latched” by a disulfide to a cysteine in β-subunit loop 1 (β1) of all vertebrate hormones except some teleost follitropins (teFSH), wherein it is latched to a cysteine in the β-subunit NH2 terminus. As reported here, teFSH analogs of human choriogonadotropin (hCG) are assembled by a pathway in which the subunits dock before the seatbelt is latched; assembly is completed by wrapping the seatbelt around loop α2 and latching it to the NH2 terminus. This differs from hCG assembly, which occurs by threading the glycosylated end of loop α2 beneath the latched seatbelt through a hole in the β-subunit. The seatbelt is the part of the β-subunit that has the greatest influence on biological function. Changes in its sequence during the divergence of lutropins, follitropins, and thyrotropins and the speciation of teleost fish may have impeded heterodimer assembly by a threading mechanism, as observed when the hCG seatbelt was replaced with its salmon FSH counterpart. Whereas wrapping is less efficient than threading, it may have facilitated natural experimentation with the composition of the seatbelt during the co-evolution of glycoprotein hormones and their receptors. Migration of the seatbelt latch site to the NH2-terminal end of the β-subunit would have facilitated teFSH assembly by a wraparound mechanism and may have contributed also to its ability to distinguish lutropin and follitropin receptors.

Influence of Subunit Interactions on Lutropin Specificity IMPLICATIONS FOR STUDIES OF GLYCOPROTEIN HORMONE FUNCTION

Bovine lutropin (bLH) and human chorionic gonadotropin (hCG) are heterodimeric glycoprotein hormones required for reproduction. Both bind rat LH receptors (rLHRs), but hCG binds human LH receptors (hLHRs) 1000-10,000 fold better than bLH. We tested the premise that this difference in affinity could be used to identify lutropin receptor contacts. Heterodimers containing hCG/bLH α- or β-subunit chimeras that bound hLHR like hCG (or bLH) were expected to have hCG (or bLH) residues at the receptor contact sites. Analogs containing one subunit derived from hCG bound hLHR much more like hCG than bLH, indicating that each bLH subunit contains all the residues sufficient for high affinity hLHR binding. Indeed, the presence of bovine α-subunit residues increased the activities of some hCG analogs. The low hLHR activity of bLH was due primarily to an interaction between its α-subunit and β-subunit residue Leu95. Leu95 does not appear to contact the hLHR since it did not influence the hLHR activity of heterodimers containing human α-subunit. These observations show that interactions within and between the subunits can significantly influence the activities of lutropins, thereby confounding efforts to identify ligand residues that contact these receptors.

Glycoprotein Hormone Assembly in the Endoplasmic Reticulum II. MULTIPLE ROLES OF A REDOX SENSITIVE β-SUBUNIT DISULFIDE SWITCH

All three human glycoprotein hormone heterodimers are assembled in the endoplasmic reticulum by threading the glycosylated end of α-subunit loop two (α2) beneath a disulfide “latched” strand of the β-subunit known as the “seatbelt.” This remarkable event occurs efficiently even though the seatbelt effectively blocks the reverse process, thereby stabilizing each heterodimer. Studies described here show that assembly is facilitated by the formation, disruption, and reformation of a loop within the seatbelt that is stabilized by the most easily reduced disulfide in the free β-subunit. We refer to this disulfide as the “tensor” because it shortens the seatbelt, thereby securing the heterodimer. Formation of the tensor disulfide appears to precede and facilitate seatbelt latching in most human choriogonadotropin β-subunit molecules. Subsequent disruption of the tensor disulfide elongates the seatbelt, thereby increasing the space beneath the seatbelt and the β-subunit core. This permits the formation of hydrogen bonds between backbone atoms of the β-subunit cystine knot and the tensor loop with backbone atoms in loop α2, a process that causes the glycosylated end of loop α2tobe threaded between the seatbelt and the β-subunit core. Contacts between the tensor loop and loop α2 promote reformation of the tensor disulfide, which explains why it is more stable in the heterodimer than in the uncombined β-subunit. These findings unravel the puzzling nature of how a threading mechanism can be used in the endoplasmic reticulum to assemble glycoprotein hormones that have essential roles in vertebrate reproduction and thyroid function.

Follitropin receptors contain cryptic ligand binding sites

Human choriogonadotropin (hCG) and follitropin (hFSH) have been shown to contact different regions of the extracellular domains of G-protein coupled lutropin (LHR) and follitropin (FSHR) receptors. We report here that hCG and hFSH analogs interact with different regions of an FSHR/LHR chimera having only two unique LHR residues and that binds both hormones with high affinity. hCG and hFSH analogs dock with this receptor chimera in a manner similar to that in which they bind LHR and FSHR, respectively. This shows that although the FSHR does not normally bind hCG, it contains a cryptic lutropin binding site that has the potential to recognize hCG in a manner similar to the LHR. The presence of this cryptic site may explain why equine lutropins bind many mammalian FSHR and why mutations in the transmembrane domain distant from the extracellular domain enable the FSHR to bind hCG. The leucine-rich repeat domain (LRD) of the FSHR also appears to contain a cryptic FSH binding site that is obscured by other parts of the extracellular domain. This will explain why contacts seen in crystals of hFSH complexed with an LRD fragment of the human FSHR are hard to reconcile with the abilities of FSH analogs to interact with membrane G-protein coupled FSHR. We speculate that cryptic lutropin binding sites in the FSHR, which are also likely to be present in thyrotropin receptors (TSHR), permit the physiological regulation of ligand binding specificity. Cryptic FSH binding sites in the LRD may enable alternate spliced forms of the FSHR to interact with FSH.

Crosslinked bifunctional gonadotropin analogs with reduced efficacy.

The N-linked oligosaccharides on human choriogonadotropin (hCG) and follitropin (hFSH) alpha-subunit loop 2 (alpha2) have a dominant influence on hormone efficacy. hCG analogs lacking this oligosaccharide retain approximately 40% the efficacy of the fully glycosylated hormone in cyclic AMP accumulation assays. Previous efforts to reduce efficacy further have involved removing the other N-linked oligosaccharides. We found that some intersubunit disulfide crosslinks reduced the efficacies of hCG analogs lacking only the alpha2 oligosaccharide. The least active analog was an hCG/hFSH chimera containing hFSH residues 95-108 in place of hCG residues 101-114 and a disulfide bond between alpha-subunit residue 37 and beta-subunit residue 33. While it bound lutropin receptors 2- to 3-fold better than hCG and follitropin receptors 10-30% as well as hFSH, it had less than 10% and 5% the efficacies of either hormone. This suggests that complete deglycosylation will not be required to produce glycoprotein hormone analogs that have low efficacies.

Partial Restoration Of Lutropin Activity by an Intersubunit Disulfide Bond: Implications For Structure/Function Studies

Gonadal function is controlled by lutropins and follitropins, heterodimeric cystine knot proteins that have nearly identical α-subunits. These heterodimeric proteins are stabilized by a portion of the hormone-specific β-subunit termed the “seatbelt” that is wrapped around α-subunit loop 2 (α2). Here we show that replacing human chorionic gonadotropin (hCG) α2 residue Lys51 with cysteine or alanine nearly abolished its lutropin activity, an observation that implies that αLys51 has a key role in hormone activity. The activity of the heterodimer containing αK51C, but not that containing αK51 A, was increased substantially when β-subunit seatbelt residue pAsp99 was converted to cysteine. As had been reported by others, heterodimers containing αK51C and βD99C were crosslinked by a disulfide. The finding that an intersubunit disulfide restored some of the activity lost by replacing αLys51 suggests that this residue is not crucial for receptor binding or signaling and also that hCG and related hormones may be particularly sensitive to mutations that alter interactions between their subunits. We propose the unique structures of hCG and related family members may permit some subunit movement in the heterodimer, making it difficult to deduce key residues involved in receptor contacts simply by correlating the activities of hormone analogs with their amino acid sequences.

Excretion of RNA catabolites by rats with hepatoma transplants

Rats with transplants of Morris Hepatoma 5123 excreted in their urine greater than normal amounts of modified nucleosides and bases, catabolites of RNA. Despite rapid growth of the neoplasm, the elevated levels did not appear until 22 days after inoculation with the tumor. With tumor progression, there were increased levels and number of these catabolites. This study also suggests that the source of the elevated RNA catabolites is mainly from the host RNA rather than from tumor tissue and also that mRNA and rRNA as well as tRNA may contribute to the urinary levels.