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• W1992872235 abstract "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. 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. Gonadotropins and thyrotropins are structurally related glycoprotein hormone heterodimers in which a loop of their α-subunits is surrounded by a strand of their β-subunits like a “seatbelt” (1Lapthorn A.J. Harris D.C. Littlejohn A. Lustbader J.W. Canfield R.E. Machin K.J. Morgan F.J. Isaacs N.W. Nature. 1994; 369: 455-461Crossref PubMed Scopus (819) Google Scholar, 2Wu H. Lustbader J.W. Liu Y. Canfield R.E. Hendrickson W.A. Structure. 1994; 2: 545-558Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 3Fox K.M. Dias J.A. Van Roey P. Mol. Endocrinol. 2001; 15: 378-389Crossref PubMed Scopus (179) Google Scholar). With the exception of some teleost fish follitropins, the seatbelts of most vertebrate hormones is “latched” by an intrasubunit disulfide to a cysteine in the β-subunit core. The unusual structures of these heterodimers and the fact that their assembly can be studied within cells makes them useful for identifying factors that affect protein folding in the ER 1The abbreviations used are: ER, endoplasmic reticulum; α1, α2, α3, α-subunit loops 1, 2, 3; β1, β2, β3, β-subunit loops 1, 2, 3; hCG, human choriogonadotropin; hFSH, human follitropin; hTSH, human thyrotropin; teFSH, teleost FSH found in salmon and related species in which the seatbelt is latched to a cysteine in the NH2-terminal end of the β-subunit. Abbreviations for all analogs are described in Fig. 1.1The abbreviations used are: ER, endoplasmic reticulum; α1, α2, α3, α-subunit loops 1, 2, 3; β1, β2, β3, β-subunit loops 1, 2, 3; hCG, human choriogonadotropin; hFSH, human follitropin; hTSH, human thyrotropin; teFSH, teleost FSH found in salmon and related species in which the seatbelt is latched to a cysteine in the NH2-terminal end of the β-subunit. Abbreviations for all analogs are described in Fig. 1. (4Ruddon R.W. Sherman S.A. Bedows E. Protein Sci. 1996; 8: 1443-1452Crossref Scopus (69) Google Scholar, 17Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35426-35436Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 18Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35437-35448Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 19Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35449-35457Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Assembly of most hCG, hFSH, and hTSH in the ER occurs after the seatbelt is latched by a process in which the glycosylated end of loop α2 is “threaded” through a hole in the β-subunit (17Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35426-35436Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). This process is facilitated by disruption of a disulfide that we have termed the “tensor” because it stabilizes a small loop within the seatbelt that regulates its length (18Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35437-35448Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Disruption of the tensor disulfide prior to assembly enlarges the hole in the β-subunit and facilitates threading; reformation of the tensor disulfide following threading tightens the seatbelt around loop α2, which stabilizes the heterodimer. Alternatively, the hCG heterodimer can be assembled by a process in which the seatbelt is wrapped around loop α2 before the seatbelt latch disulfide is formed; formation of the seatbelt latch disulfide completes assembly. The “wrapping” mechanism, which was first proposed on the basis of pulse-chase analysis (4Ruddon R.W. Sherman S.A. Bedows E. Protein Sci. 1996; 8: 1443-1452Crossref Scopus (69) Google Scholar), appears to be used infrequently relative to the threading pathway for hCG assembly. Wrapping is required for assembly of hCG analogs in which the seatbelt is latched to a cysteine in the NH2-terminal end of the β-subunit, a site comparable with that of the FSH β-subunit found in salmon and many other teleost fish (19Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35449-35457Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). It is also required for the assembly of heterodimers in which the seatbelt is latched to the α-subunit (5Xing Y. Lin W. Jiang M. Myers R.V. Cao D. Bernard M.P. Moyle W.R. J. Biol. Chem. 2001; 276: 46953-46960Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar).Studies described here were designed to learn why most hCG is assembled by a threading mechanism rather than a wraparound mechanism. Conceivably, the latched seatbelt is a component of the subunit docking site. As a result, formation of the seatbelt latch disulfide would increase the affinity of the β-subunit for the α-subunit, thereby facilitating assembly by a threading route. Alternatively, the unlatched hCG seatbelt might occupy positions near the subunit interface where it would be in a position to interfere with subunit docking or where it can destabilize the docked complex. This would reduce the amount of docked complex and interfere with assembly by a wraparound route. The finding that the end of the seatbelt scans the β-subunit to find its latch site (18Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35437-35448Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) supports the notion that the seatbelt could contact the subunit interface. Efforts to distinguish these possibilities led us to study how the hCG α- and β-subunits dock and to determine how the latching of the seatbelt affected this process.Using intersubunit disulfide bonds to stabilize partially assembled intermediates, we found that the hCG β-subunit docks with the α-subunit in similar but not identical fashions when its seatbelt is latched or unlatched. Most docked complexes appear to dissociate before the heterodimer can be assembled by either pathway, a phenomenon that would favor threading by providing more time for the β-subunit to latch its seatbelt. Furthermore, the unlatched seatbelt appears to hinder docking and/or promote dissociation of the docked complex, a phenomenon that would also favor threading. By comparing the apparent positions of the subunits in the docked complexes with the structures of the assembled heterodimers, we devised models of heterodimer assembly. These suggest that while both threading and wrapping depend on the formation of similar intrasubunit and intersubunit hydrogen bonds, these would appear to form more readily by threading when the seatbelt is latched than by wrapping when it is unlatched.EXPERIMENTAL PROCEDURESConstructs used in these studies are illustrated in Fig. 1 and were produced by standard methods of site-directed mutagenesis (17Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35426-35436Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Methods used to transfect COS-7 cells and immunological procedures employed to measure the resulting heterodimers have also been described (17Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35426-35436Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). To facilitate identification of the analogs used in each of the studies described here, we named them to reflect the hCG residues that have been changed. For example, hCGβ-R8C,C93A,C100A represents an analog of the hCG β-subunit in which codons for Arg8, Cys93, and Cys100 were replaced with cysteine, alanine, and alanine, respectively. The relative locations of antibody binding sites used in the hormone sandwich immunoassays are illustrated by Xing et al. (17Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35426-35436Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Briefly, most heterodimers were captured to microtiter plates using an antibody (A113) to the α-subunit and detected using a radioiodinated antibody (B110 or B111) to the β-subunit. Molecular modeling and cartoon illustrations were prepared with the aid of the programs Sybyl (Tripos, St. Louis, MO) and Sculpt (MDL Information Systems, Inc., San Diego, CA).RESULTSThe Subunits Dock in Similar but Not Identical Fashions When the Seatbelt Is Latched and Unlatched—We employed a disulfide scanning mutagenesis strategy to identify portions of the subunits that are likely to contact one another when the seatbelt is unlatched or when the tensor disulfide is disrupted. These are key intermediates in the wraparound and threading pathways, respectively (17Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35426-35436Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The analogs used were derivatives of β-subunits that cannot latch their seatbelts (i.e. hCGβ-C26A,C110A) or form the tensor disulfide (i.e. hCGβ-C93A,C100A). While neither was capable of being assembled into stable heterodimers with the native α-subunit (6Suganuma N. Matzuk M.M. Boime I. J. Biol. Chem. 1989; 264: 19302-19307Abstract Full Text PDF PubMed Google Scholar), both were incorporated into heterodimers that are cross-linked by an intersubunit disulfide (Fig. 2). This property formed the basis of our strategy for identifying portions of the subunits likely to contact one another during assembly. We assumed that the ability of a disulfide to “rescue” complexes containing docked subunits that cannot otherwise be assembled into a heterodimer would be proportional to the time that its component cysteines are adjacent. Based on our expectation that contacts between the α-subunit and these β-subunit analogs during wrapping and threading would be at least somewhat similar to those in the heterodimer, we introduced cysteines into each subunit at sites that were capable of cross-linking the heterodimer. This approach may have caused us to overlook transient contacts that do not lead to assembly, but these were of lesser interest for these studies.Several intersubunit disulfides have been used to cross-link the subunits of hCG (7Heikoop J.C. van den Boogaart P. Mulders J.W.M. Grootenhuis P.D.J. Nat. Biotech. 1997; 15: 658-662Crossref PubMed Scopus (46) Google Scholar, 8Einstein M. Lin W. Macdonald G.J. Moyle W.R. Exp. Biol. Med. 2001; 226: 581-590Crossref Scopus (12) Google Scholar) and we selected a few others by measuring the distances between the Cα carbons and between the Cβ carbons of every residue in the α- and β-subunits (not shown). We assumed that the most favorable disulfides would be at positions in which the maximum distances between these atoms would be ∼6–6.5 and 3.8–4.2 Å, respectively. Based on these considerations, we substituted cysteines for residues in the hCG α- and β-subunits to produce heterodimers having the potential to form the following disulfides: αQ5C-βR8C, αQ27C-βV44C, αV76C-βV44C, αC7S-βY37C, αR35C-βA35C, αY37C-βI33C, and αK51C-βD99C (Fig. 1). Note that the conversion of αCys7 to serine in α-C7S disrupted its ability to form an intra-subunit disulfide between α-subunit residues 7 and 31. The resulting free α-subunit cysteine (i.e. αCys31) can form a disulfide with the cysteine introduced into the β-subunit in place of hCG-βTyr37 (8Einstein M. Lin W. Macdonald G.J. Moyle W.R. Exp. Biol. Med. 2001; 226: 581-590Crossref Scopus (12) Google Scholar). As described next, each of these analogs formed cross-linked heterodimers that were stable at pH 2, 37 °C, a condition known to promote hCG dissociation (9Aloj S.M. Edelhock H. Ingham K.C. Morgan F.J. Canfield R.E. Ross G.T. Arch. Biochem. Biophys. 1973; 159: 497-504Crossref PubMed Scopus (47) Google Scholar). Formation of these disulfides in complexes lacking the abilities to latch their seatbelts or to stabilize the small seatbelt loop indicated that these portions of the subunits were also adjacent at some time during the period in which the subunits were docked to one another.Fig. 1Constructs used in these studies. The amino acid sequences of the constructs used in these studies are shown here. Residues above the sequence indicate amino acid substitutions. For example, α-Q5C represents an analog in which α-subunit residue αGln5 is converted to cysteine. Several analogs contain two or more substitutions. For example, hCGβ-C26A,C110A represents an analog in which both βCys26 and βCys110 are converted to alanine. As indicated by the bracket, hCGβ-δ(93:100)DA represents an analog in which all the residues in the tensor loop are replaced by aspartic acid and alanine. hCGβ-δ(93:100)DA,C26A is an analog of hCGβ-δ(93:100)DA in which βCys26 is converted to alanine, a mutation that prevents it from latching its seatbelt to loop β1. δ1,7hCGβ refers to an analog lacking residues 1–7 and that has an arginine at its N terminus (i.e. corresponding to hCG βArg8). δ2,8hCGβ is a construct encoding an hCGβ analog missing residues 2–8. This analog has a serine at its NH2 terminus (i.e. corresponding to hCG βSer1).View Large Image Figure ViewerDownload (PPT)To learn how interactions between the NH2-terminal portions of the subunits might affect subunit docking, we studied the formation of analogs that were cross-linked by the α5-β8 disulfide. Co-expression of α-Q5C and hCGβ-R8C led to the formation of an acid-stable heterodimer, indicating that it contained an intersubunit disulfide. When α-Q5C was co-expressed with hCGβ-R8C,C26A,C110A and hCGβ-R8C,C93A, C100A, β-subunits that cannot latch their seatbelts or form their tensor disulfides, respectively, we observed that 125 and 39% as much heterodimer was formed as that containing hCGβ-R8C (Fig. 2, left and right). This showed that an intersubunit disulfide had formed between the NH2-terminal ends of the α- and β-subunits while the subunits were docked with one another, even though the seatbelt latch and tensor disulfides could not be formed. It also revealed that the NH2-terminal portions of both types of β-subunits appear to contact the NH2-terminal portions of the α-subunit during subunit docking. The relative differences in the amounts of cross-linked docked subunits observed are impossible to interpret, however. They might indicate that contacts between the NH2-terminal ends of the subunits are favored more when the seatbelt is unlatched than when the tensor disulfide is disrupted. Then again, they might also reflect the tendency of seatbelt residue βCys110 to form a disulfide with βCys8 in hCGβ-R8C,C93A,C100A (18Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35437-35448Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), which would have rendered βCys8 incapable of forming an intersubunit disulfide with αCys5. Consequently, only that fraction of hCGβ-R8C,C93A,C100A in which the seatbelt is latched to βCys26 would be capable of being cross-linked by a disulfide.Parts of loop β2 contact loops α1 and α3 in hCG (1Lapthorn A.J. Harris D.C. Littlejohn A. Lustbader J.W. Canfield R.E. Machin K.J. Morgan F.J. Isaacs N.W. Nature. 1994; 369: 455-461Crossref PubMed Scopus (819) Google Scholar, 2Wu H. Lustbader J.W. Liu Y. Canfield R.E. Hendrickson W.A. Structure. 1994; 2: 545-558Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar) and in hFSH (3Fox K.M. Dias J.A. Van Roey P. Mol. Endocrinol. 2001; 15: 378-389Crossref PubMed Scopus (179) Google Scholar). To learn if these portions of the hCG β-subunit might participate in docking, we tested the abilities of cysteines that had been introduced in place of loop α1 residue αQ27C and loop α3 residue αV76C to form intersubunit disulfides with β-subunit analogs that contain a cysteine in place of hCG loop β2 residue βV44C. Co-expression of α-Q27C or α-V76C with hCGβ-V44C led to the formation of cross-linked heterodimers, all of which were acid stable. The α27-β44 and α76-β44 disulfides each stabilized 28% of the complexes containing the β-subunit that cannot latch its seatbelt. They were not as effective as the disulfide in the subunit NH2 termini (Fig. 2, left). This suggested that when the hCG seatbelt is unlatched, the subunits dock in orientations that favor the formation of NH2-terminal contacts relative to those between loop β2 and loops α1 and α3.The α27-β44 disulfide also rescued heterodimers that are unable to form the tensor disulfide. As a result, co-expression of α-Q27C and hCGβ-V44C,C93A,C100A led to 54% as much heterodimer that was formed when α-Q27C was expressed with hCGβ-V44C (Fig. 2, right). This was as good or better than the α5-β8 disulfide (i.e. 39%). Remarkably, the α76-β44 disulfide between αV76C in loop α3 and βV44C in loop β2 did not rescue docked complexes containing hCGβ-V44C,C93A,C100A (Fig. 2, right), an observation that remains puzzling. We did not expect to find that the α27-β44 disulfide would rescue docked complexes containing β-subunits that cannot form their tensor disulfides better than those that cannot latch their seatbelts. This was because hCGβ-V44C,C93A,C100A has the potential to latch its seatbelt to βCys44 and, as a consequence, this cysteine would not be capable of being cross-linked to residue αCys27 in α-Q27C. In contrast, it is not possible for hCGβ-C26A,V44C,C110A to latch its seatbelt to βCys44. Thus, the lower ability of the α27-β44 disulfide to rescue docked complexes containing an unlatched seatbelt may indicate that before it is latched, the seatbelt may impede the formation of contacts between the loops α1 and α3 with loop β2.We studied potential contacts between loop α2 and either the cystine knot or loop β1 using analogs that can form intersubunit disulfide bonds between residues α35-β35 and α37-β33. Both disulfides have been shown to cross-link the subunits in hCG (7Heikoop J.C. van den Boogaart P. Mulders J.W.M. Grootenhuis P.D.J. Nat. Biotech. 1997; 15: 658-662Crossref PubMed Scopus (46) Google Scholar). Each rescued docked complexes containing β-subunits having latched seatbelts and disrupted tensor disulfides better than complexes containing β-subunits with unlatched seatbelts and intact tensor disulfides. Thus, the α35-β35 disulfide rescued 66% of the heterodimer containing α-R35C and hCGβ-A35C,C93A,C100A (Fig. 2, right), but only 8.5% of the heterodimer containing α-R35C and hCGβ-C26A,A35C,C110A (Fig. 2, left). The α37-β33 disulfide rescued 25% of the heterodimer containing α-Y37C and hCGβ-I33C,C93A,C100A (Fig. 2, right), but only 3.9% of the heterodimer containing α-Y37C and hCGβ-C26A,I33C,C110A (Fig. 2, left). The observations that the α35-β35 and α37-β33 disulfides formed more readily in analogs of hCGβ-C93A,C100A, which have latched seatbelts and disrupted tensor disulfides (Fig. 2, right), than in analogs of hCGβ-C26A,C110A, which have unlatched seatbelts and intact tensor disulfides (Fig. 2, left), suggested that loop α2 residues αArg35 and αTyr37 are more highly constrained during threading than wrapping. This would lead to increased contacts between the subunits, which would be expected to increase the stability of the docked complex.Efforts to detect other potential contacts that might stabilize the docked complex prior to assembly by the wraparound pathway led us to test the ability of the α31-β37 disulfide to secure the heterodimer. This disulfide was found to stabilize an hCG analog formed by co-expressing hCGβ-Y37C with α-C7A (8Einstein M. Lin W. Macdonald G.J. Moyle W.R. Exp. Biol. Med. 2001; 226: 581-590Crossref Scopus (12) Google Scholar) and was observed to rescue 15.6% of this material when hCGβ-C26A,Y37C,C110A was co-expressed with α-C7S (Fig. 2, right). We did not repeat these studies with analogs that are unable to form the tensor loop, because disulfides on either side of α31-β37 (i.e. α27-β44, α35-β35, and α37-β33) had already been found to rescue a much larger fraction of the heterodimer (Fig. 2, right). Considered together, these studies indicated that contacts near the cystine knots were likely to make a greater contribution to the threading pathway than to the wraparound pathway. The finding that these areas of the subunits are less likely to contact one another when the seatbelt is unlatched may reflect the ability of the unlatched seatbelt to disrupt contacts between the subunits, a topic to be considered later.We anticipated that the seatbelt would make extensive contacts with residues in loop α2 during the threading pathway and that we would not be able to distinguish these from contacts made during docking. Indeed, we had already found that disulfide bonds appear to form between unpaired cysteines in loop α2 and the tensor disulfides during threading (18Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35437-35448Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). To identify contacts between the seatbelt and the α-subunit that might facilitate docking in the wraparound pathway, we took advantage of a disulfide that forms between the seatbelt and loop α2, i.e. α51-β99 (7Heikoop J.C. van den Boogaart P. Mulders J.W.M. Grootenhuis P.D.J. Nat. Biotech. 1997; 15: 658-662Crossref PubMed Scopus (46) Google Scholar, 8Einstein M. Lin W. Macdonald G.J. Moyle W.R. Exp. Biol. Med. 2001; 226: 581-590Crossref Scopus (12) Google Scholar). This disulfide rescued heterodimers containing α-K51C with hCGβ-C26A,D99C,C110A to 25.8% of the level observed when α-K51C was co-expressed with hCGβ-D99C (Fig. 2, left). This suggested that the tensor loop can participate in contacts with loop α2 while the seatbelt is unlatched. As noted later, we anticipate that hydrogen bonds between the backbone atoms of loop α2 residues αVal53-αGlu56 and seatbelt residues βThr98-βGly101, which include part of the tensor loop, are necessary for efficient completion of assembly by the wraparound pathway. These contacts do not appear to stabilize NH2-terminal portions of loop α2 that contact the β-subunit cystine knot, however, because analogs that are unable to latch their seatbelts were not rescued effectively by the α31-β37, α35-β35, or α37-β33 disulfides (Fig. 2, left).The abilities of intersubunit disulfides to rescue docked complexes containing β-subunits that cannot latch their seatbelts or form the tensor disulfide suggest that the manner in which subunits dock is similar but nonidentical during assembly by threading and wrapping mechanisms. The finding that the α5-β8 disulfide rescued the formation of both types of heterodimers supported the notion that contacts in the NH2-terminal region are important for the assembly of hCG and other lutropins. This is consistent with the observation that deletion of residues in the NH2 terminus of the β-subunit reduced secretion of hCG analogs in which the seatbelt is latched normally (10Huang J. Chen F. Puett D. J. Biol. Chem. 1993; 268: 9311-9315Abstract Full Text PDF PubMed Google Scholar, 11Slaughter S. Wang Y.H. Myers R.V. Moyle W.R. Mol. Cell. Endocrinol. 1995; 112: 21-25Crossref PubMed Scopus (17) Google Scholar).The ability of the α5-β8 disulfide to stabilize heterodimers lacking the abilities to latch their seatbelts suggested that contacts between the NH2-terminal portions of the subunits may have a dominant role during assembly by the wraparound pathway. To test this possibility, we monitored the abilities of analogs to form cross-linked heterodimers during assembly that can occur only by the wraparound pathway (Table I). Elimination of residues 1–7 or 2–8 reduced heterodimer secretion substantially (Table I). They also reduced assembly of heterodimers in which the seatbelt is latched to either α-subunit residue 37 (Table I, study 1) or to α-subunit residue 43 (Table I, study 2). This suggests that NH2-terminal contacts are likely to have a role in subunit docking even though they are not essential for docking.Table IInfluence of NH2 terminal hCGβ residues 1–7 and 2–8 on heterodimer assembly by threading and wraparound pathwaysData rowα-Subunitβ-SubunitTotal heterodimerng/50 μll ± S.E.Study 1, seatbelt latched to β26 or α371NativehCGβ14.73 ± 0.052Nativeδ1,7-hCGβ1.30 ± 0.023α-Y37ChCGβ-C26A5.92 ± 0.234α-Y37Cδ1,7-hCGβ-C26A<0.1Study 2, seatbelt latched to β26 or α435NativehCGβ27.75 ± 2.936Nativeδ1,7-hCGβ1.64 ± 0.097Nativeδ2,8-hCGβ<0.18α-S43ChCGβ-C26A7.56 ± 0.109α-S43Cδ1,7-hCGβ-C26A2.04 ± 0.13 Open table in a new tab The contribution of NH2-terminal contacts to assembly may explain our inability to detect assembly of hTSH and hFSH by a wraparound mechanism. These β-subunits have only one and two residues in their NH2-terminal ends, respectively. Therefore, the finding that hTSH and hFSH were unable to form heterodimers by a wraparound mechanism (17Xing Y. Myers R.V. Cao D. Lin W. Jiang M. Bernard M.P. Moyle W.R. J. Biol. Chem. 2004; 279: 35426-35436Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) may indicate that contacts between the NH2-terminal portions of both subunits are more important for assembly by a wrapping mechanism than for assembly by threading.Docking Is Readily Reversible, a Phenomenon That May Delay Most hCG Assembly Until the Seatbelt Is Latched—Experiments described next were performed during efforts to identify rate-limiting steps in glycoprotein hormone assembly and thereby learn why most hCG is assembled by a threading mechanism. These studies depended on our abilities to identify the relative rates of subunit docking, threading, and wrapping in the ER. We monitored these processes using the α5-β8 disulfide because of its ability to trap and rescue docked complexes in which the seatbelt is unlatched, a phenomenon that was required to detect early stages in the wraparound pathway. During these studies we compared the abilities of hCGβ to compete with hCGβ-R8C and hCGβ-R8C,C26A,C110A for α-Q5C for heterodimer formation. We reasoned that if threading or wrapping were not rate-limiting and occurred immediately after the subunits dock, then hCGβ would compete efficiently with hCGβ-R8C or hCGβ-R8C,C26A,C110A for heterodimer formation. Consequently, a substantial fraction of the heterodimer formed in the presence of hCGβ would lack an intersubunit disulfide and dissociate at pH 2, 37 °C. In contrast, if the subunits docked and undocked faster than the heterodimer became stabilized by threading or wrapping mechanisms, then hCGβ-R8C and hCGβ-R8C,C26A,C110A would constitute most of the β-subunit in the heterodimer. This is because forma" @default.
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