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

Abstract

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...

N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

“…Cell Culture and Transfections-The HEK293 i -Mych␦OR-FLAG cell line has been described elsewhere (18,19). The HEK293 i -Myc-h␦OR(N18Q)-FLAG, HEK293 i -Mych␦OR(N33Q)-FLAG, and HEK293 i -Myc-h␦OR(N18Q/N33Q)-FLAG cell lines were created with the same protocol under blasticidin S (4 g/ml; InVivogen) and hygromycin (400 g/ml; InVivogen) selection.…”

“…The enzymes were added to a final concentration of 50 milliunits/ml, and samples were incubated at 30°C for 16 h before terminating the reaction by adding 40 l of SDS-sample buffer. Endo H and PNGase F digestions were performed as described (19).…”

Alternative splicing of G protein-coupled receptors: physiology and pathophysiology

“…The male patient had delayed puberty, micropenis and oligospermia; while two of his sisters were infertile and the third sister had three spontaneous miscarriages [53]. Another splice variant of LHR that results in a frame-shift in the reading frame and creates a truncated receptor composed of the N-terminal ectodomain (responsible for high-affinity ligand binding), causes an alteration in pituitary-gonadal function, and morphological changes in the adrenals and kidneys of transgenic mice [54].…”

“…This variant also appears to control the cell surface expression of the wild-type receptor by misrouting newly synthesized receptors in the ER [54].…”

Cys-27 Variant of Human δ-Opioid Receptor Modulates Maturation and Cell Surface Delivery of Phe-27 Variant via Heteromerization

“…In support of this idea are the observations for an increasing number of naturally occurring GPCR slice variants and mutant forms as well as engineered receptor mutants that have been shown to behave as dominant-negatives of their corresponding wild-type forms by preventing their expression at the cell surface (e.g. rhodopsin (22) and D3 dopamine (23), D2 dopamine (24), gonadotropin-releasing hormone (25,26), melanocortin-1 (27,28), thyroid-stimulating hormone (29), luteinizing hormone (30), CCR5 chemokine (31,32), and V2 vasopressin (33) receptors). On the other hand, a few family A GPCRs have been shown to promote cell surface delivery of other receptors.…”