Rescue of Intracellularly Trapped Lutropin Receptor Exodomain by Endodomain and Reconstitution of a Functional Membrane Receptor: Interaction between Exo- and Endodomains

Bozon, Véronique; Couture, Laurence; Pajot‐Augy, Edith; Richard, Fabien; Rémy, Jean-Serge; Salesse, Roland

doi:10.1006/prep.2002.1617

Cited by 10 publications

(6 citation statements)

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“…[11] first reported the production and secretion of soluble LH receptor following transient transfection of a naturally truncated variant of the receptor in COS cells. These data were independently substantiated using different combinations of in vitro expression systems [12,13], In some studies, the lack of detection of an actively secreted soluble form of expressed LH receptor was attributed to misfolding and intra-cellular retention of the expressed protein or proteolytic degradation of the protein released into the culture media [13,14]. Moreover, the lack of a secreted form of the receptor was overcome by co-expression of hCG with the extra-cellular domain of porcine LH receptor leading to the secretion of a mixture of free as well as hCG-receptor complex [13].…”

Section: Introductionmentioning

confidence: 99%

Microvesicle-mediated release of soluble LH/hCG receptor (LHCGR) from transfected cells and placenta explants

Chambers

Stanley

Randeva

et al. 2011

Reprod Biol Endocrinol

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Placental hCG and pitutary LH transduce signals in target tissues through a common receptor (LHCGR). We demonstrate that recombinant LHCGR proteins which include the hormone-binding domain are secreted from transfected cells and that natural LHCGR is also secreted from human placental explants. LHCGR recombinant proteins representing varying lengths of the N-terminal extracellular domain were expressed in Chinese Hamster Ovary cells in suspension culture. Secretion was minimal up to 72h but by 96h 24-37% of the LHCGR had been released into the culture medium. The secreted proteins were folded and sensitive to glycosidases suggesting N-linked glycosylation. Secretion was independent of recombinant size and was mediated via structurally defined membrane vesicles (50-150nm). Similarly cultured human early pregnancy placental explants also released LHCGR via microvesicles. These studies provide the first experimental evidence of the possible mechanistic basis of the secretion of LHCGR.

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Section: Introductionmentioning

confidence: 99%

Microvesicle-mediated release of soluble LH/hCG receptor (LHCGR) from transfected cells and placenta explants

Chambers

Stanley

Randeva

et al. 2011

Reprod Biol Endocrinol

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“…The cysteines of C-b2 are linked to cysteines of C-b3 close to TMH1 by disulfide bonds (16). Indirect evidence permits the global assignment of disulfide bridges between the six cysteines (17)(18)(19)(20). Especially two recent publications on TSHR (21) and LHCGR (13) suggest disulfide bridges between Cys 283 (C-b2)/ Cys 398 (C-b3) and Cys 284 (C-b2)/Cys 408 (C-b3), whereas Cys 301 (in C-b2) is likely to be paired with Cys 390 (in C-b3).…”

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confidence: 99%

Defining Structural and Functional Dimensions of the Extracellular Thyrotropin Receptor Region

Kleinau

Mueller

Jaeschke

et al. 2011

Journal of Biological Chemistry

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The extracellular region of the thyrotropin receptor (TSHR) can be subdivided into the leucine-rich repeat domain (LRRD) and the hinge region. Both the LRRD and the hinge region interact with thyrotropin (TSH) or autoantibodies. Structural data for the TSHR LRRD were previously determined by crystallization (amino acids Glu 30 -Thr 257 , 10 repeats), but the structure of the hinge region is still undefined. Of note, the amino acid sequence (Trp 258 -Tyr 279 ) following the crystallized LRRD comprises a pattern typical for leucine-rich repeats with conserved hydrophobic side chains stabilizing the repeat fold. Moreover, functional data for amino acids between the LRRD and the transmembrane domain were fragmentary. We therefore investigated systematically these TSHR regions by mutagenesis to reveal insights into their functional contribution and potential structural features. We found that mutations of conserved hydrophobic residues between Thr 257 and Tyr 279 cause TSHR misfold, which supports a structural fold of this peptide, probably as an additional leucine-rich repeat. Furthermore, we identified several new mutations of hydrophilic amino acids in the entire hinge region leading to partial TSHR inactivation, indicating that these positions are important for intramolecular signal transduction. In summary, we provide new information regarding the structural features and functionalities of extracellular TSHR regions. Based on these insights and in context with previous results, we suggest an extracellular activation mechanism that supports an intramolecular agonistic unit as a central switch for activating effects at the extracellular region toward the serpentine domain.

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“…However, the 22-amino acid C-terminal tail of the variant contains an additional cysteine ( Figure 1B) that might interfere with the formation of the correct disulfide bonds. Interestingly, when the truncated ectodomain is coexpressed with the C-terminal portion of the receptor containing the transmembrane and cytoplasmic domains, they form a functional receptor that is delivered to the cell surface (Bozon et al, 2002). In contrast, coexpression of the extracellular variant and the full-length receptor did not lead to secretion of the variant but resulted in the intracellular retention of the receptor.…”

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Luteinizing Hormone Receptor Ectodomain Splice Variant Misroutes the Full-Length Receptor into a Subcompartment of the Endoplasmic Reticulum

Apaja

Tuusa

Pietilä

et al. 2006

MBoC

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The luteinizing hormone receptor (LHR) is a G protein-coupled receptor that is expressed in multiple RNA messenger forms. The common rat ectodomain splice variant is expressed concomitantly with the full-length LHR in tissues and is a truncated transcript corresponding to the partial ectodomain with a unique C-terminal end. Here we demonstrate that the variant alters the behavior of the full-length receptor by misrouting it away from the normal secretory pathway in human embryonic kidney 293 cells. The variant was expressed as two soluble forms of M r 52,000 and M r 54,000, but although the protein contains a cleavable signal sequence, no secretion to the medium was observed. Only a very small fraction of the protein was able to gain hormone-binding ability, suggesting that it is retained in the endoplasmic reticulum (ER) by its quality control due to misfolding. This was supported by the finding that the variant was found to interact with calnexin and calreticulin and accumulated together with these ER chaperones in a specialized juxtanuclear subcompartment of the ER. Only proteasomal blockade with lactacystin led to accumulation of the variant in the cytosol. Importantly, coexpression of the variant with the full-length LHR resulted in reduction in the number of receptors that were capable of hormone binding and were expressed at the cell surface and in targeting of immature receptors to the juxtanuclear ER subcompartment. Thus, the variant mediated misrouting of the newly synthesized full-length LHRs may provide a way to regulate the number of cell surface receptors. INTRODUCTIONNewly synthesized proteins destined for the secretory pathway enter the endoplasmic reticulum (ER) and after correct folding and assembly are either secreted from the cell or transported to their site of action in other cellular compartments. Folding of the nascent proteins is constantly monitored by the ER quality control apparatus. It has an important task in maintaining the cellular homeostasis by preventing premature export of incompletely folded and assembled proteins and removing misfolded and unassembled ones via the ER-associated degradation (ERAD) pathway, presumably by recognizing structural signals that are enriched in incompletely folded proteins (Ellgaard and Helenius, 2003;Helenius and Aebi, 2004). Recent evidence suggests that the ER quality control does not only target permanently misfolded proteins to ERAD but may also dispose some folding competent ones, possibly due to their slow folding kinetics. This applies also to several polytopic membrane proteins, including G protein-coupled receptors (GPCRs; Petäjä-Repo et al., 2000;Imai et al., 2001;Lu et al., 2003;Wü ller et al., 2004;Pietilä et al., 2005). Thus, export from the ER is the limiting step in their expression and probably provides a mean to regulate the number of functional receptors at the cell surface. This is supported by our recent finding that final maturation of the rat luteinizing hormone receptor (rLHR) was found to be developmentally regulated in targ...

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Rescue of Intracellularly Trapped Lutropin Receptor Exodomain by Endodomain and Reconstitution of a Functional Membrane Receptor: Interaction between Exo- and Endodomains

Cited by 10 publications

References 53 publications

Microvesicle-mediated release of soluble LH/hCG receptor (LHCGR) from transfected cells and placenta explants

Microvesicle-mediated release of soluble LH/hCG receptor (LHCGR) from transfected cells and placenta explants

Defining Structural and Functional Dimensions of the Extracellular Thyrotropin Receptor Region

Luteinizing Hormone Receptor Ectodomain Splice Variant Misroutes the Full-Length Receptor into a Subcompartment of the Endoplasmic Reticulum

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