Misfolding Ectodomain Mutations of the Lutropin Receptor Increase Efficacy of Hormone Stimulation

Abstract We demonstrate 2 novel mutations of the LHCGR, each homozygous, in a 46,XY patient with severe Leydig cell hypoplasia. One is a mutation in the signal peptide (p.Gln18_Leu19ins9; referred to here as SP) that results in an alteration of the coding sequence of the N terminus of the mature mutant receptor. The other mutation (p.G71R) is also within the ectodomain. Similar to many other inactivating mutations, the cell surface expression of recombinant human LHR(SP,G71R) is greatly reduced due to intracellular retention. However, we made the unusual discovery that the intrinsic efficacy for agonist-stimulated cAMP in the reduced numbers of receptors on the cell surface was greatly increased relative to the same low number of cell surface wild-type receptor. Remarkably, this appears to be a general attribute of misfolding mutations in the ectodomains, but not serpentine domains, of the gonadotropin receptors. These findings suggest that there must be a common, shared mechanism by which disparate mutations in the ectodomain that cause misfolding and therefore reduced cell surface expression concomitantly confer increased agonist efficacy to those receptor mutants on the cell surface. Our data further suggest that, due to their increased agonist efficacy, extremely small changes in cell surface expression of misfolded ectodomain mutants cause larger than expected alterations in the cellular response to agonist. Therefore, for inactivating LHCGR mutations causing ectodomain misfolding, the numbers of cell surface mutant receptors on fetal Leydig cells of 46,XY individuals exert a more exquisite effect on the relative severity of the clinical phenotypes than already appreciated.

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Loss-of-Function and Gain-of-Function Mutations of Calcium-Sensing Receptor: Functional Analysis and the Effect of Allosteric Modulators NPS R-568 and NPS 2143

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2013-1974 ◽

2013 ◽

Vol 98 (10) ◽

pp. E1692-E1701 ◽

Cited By ~ 15

Author(s):

Akie Nakamura ◽

Tomoyuki Hotsubo ◽

Keiji Kobayashi ◽

Hiroshi Mochizuki ◽

Katsura Ishizu ◽

...

Keyword(s):

Cell Surface ◽

Surface Expression ◽

Cell Surface Expression ◽

Allosteric Modulators ◽

Familial Hypocalciuric Hypercalcemia ◽

Calcium Sensing Receptor ◽

Activating Mutations ◽

Casr Gene ◽

Calcium Sensing ◽

Inactivating Mutations

Abstract Objective: Activating mutations in the calcium-sensing receptor (CASR) gene cause autosomal dominant hypoparathyroidism, and heterozygous inactivating CASR mutations cause familial hypocalciuric hypercalcemia. Recently, there has been a focus on the use of allosteric modulators to restore the functional activity of mutant CASRs. In this study, the effect of allosteric modulators NPS R-568 and NPS 2143 on CASR mutants was studied in vitro. Methods: DNA sequence analysis of the CASR gene was undertaken in autosomal dominant hypoparathyroidism and familial hypocalciuric hypercalcemia Japanese patients, and the functional consequences for the Gi-MAPK pathway and cell surface expression of CASR were determined. Furthermore, we studied the effect of NPS R-568 and NPS 2143 on the signal transduction activity and cell surface expression of each mutant CASR. Results: We identified 3 activating mutations (S122C, P569H, and I839T) and 2 inactivating mutations (A110T and R172G) in patients. The activating and inactivating mutations caused leftward and rightward shifts, respectively, in the dose-response curves of the signaling pathway. NPS R-568 rescued the signal transduction capacity of 2 inactivating mutants without increasing cell surface expression levels. NPS 2143 suppressed the enhanced activity of the activating mutants without altering cell surface expression levels, although A843E, which is a constitutively active mutant, was suppressed to a lesser degree. Conclusions: We have identified 4 novel mutations of CASR. Moreover, our results indicate that allosteric modulators can restore the activity of the loss- and gain-of-function mutant CASRs, identified in this study.

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Individual cells traffic the Vasopressin 2 Receptor to their cell surface with different success

10.1101/2021.08.09.455709 ◽

2021 ◽

Author(s):

Eline J Koers ◽

Bradley A Morgan ◽

Iain B Styles ◽

Dmitry J Veprintsev

Keyword(s):

Cell Surface ◽

Receptor Cell ◽

Surface Expression ◽

G Protein Coupled Receptors ◽

Cell Surface Expression ◽

Subcellular Localisation ◽

Equal Distribution ◽

Mutant Receptors ◽

G Protein Coupled ◽

Correct Expression

G protein coupled receptors (GPCRs) translate the actions of hormones and neurotransmitters into intracellular signalling events. Mutations in GPCRs can prevent their correct expression and trafficking to the cell surface and cause disease. Single cell subcellular localisation measurements reveal that while some cells appear to traffic the majority of the vasopressin 2 receptor (V2R) molecules to the cell surface, others retain a greater number of receptors in the ER or have approximately equal distribution. Mutations in the V2R affect the proportion of cells able to send this GPCR to their cell surface but surprisingly they do not prevent all cells from correctly trafficking the mutant receptors. These findings reveal the potential for rescue of mutant receptor cell surface expression by pharmacological manipulation of the GPCR folding and trafficking machinery.

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Cell density-dependent regulation of cell surface expression of two types of human tumor necrosis factor receptors and its effect on cellular response

Journal of Cellular Biochemistry ◽

10.1002/jcb.240540412 ◽

1994 ◽

Vol 54 (4) ◽

pp. 453-464 ◽

Cited By ~ 16

Author(s):

Eva Pocsik ◽

Rudolf Mihalik ◽

Francis Ali-Osman ◽

Bharat B. Aggarwal

Keyword(s):

Tumor Necrosis Factor ◽

Cell Surface ◽

Cellular Response ◽

Tumor Necrosis ◽

Human Tumor ◽

Surface Expression ◽

Cell Surface Expression ◽

Human Tumor Necrosis Factor ◽

Tumor Necrosis Factor Receptors ◽

Necrosis Factor

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A Point Mutation in the Human Melanin Concentrating Hormone Receptor 1 Reveals an Important Domain for Cellular Trafficking

Molecular Endocrinology ◽

10.1210/me.2004-0301 ◽

2005 ◽

Vol 19 (10) ◽

pp. 2579-2590 ◽

Cited By ~ 48

Author(s):

Jun Fan ◽

Stephen J. Perry ◽

Yinghong Gao ◽

David A. Schwarz ◽

Richard A. Maki

Keyword(s):

Endoplasmic Reticulum ◽

Cell Surface ◽

Point Mutation ◽

Hormone Receptor ◽

Surface Expression ◽

Cell Surface Expression ◽

Single Mutation ◽

Wild Type ◽

Melanin Concentrating Hormone ◽

Type Receptor

Abstract G protein-coupled receptors (GPCRs) are heptahelical integral membrane proteins that require cell surface expression to elicit their effects. The lack of appropriate expression of GPCRs may be the underlying cause of a number of inherited disorders. There is evidence that newly synthesized GPCRs must attain a specific conformation for their correct trafficking to the cell surface. In this study, we show that a single point mutation in human melanin-concentrating hormone receptor (hMCHR1) at position 255 (T255A), which is located at the junction of intracellular loop 3 and transmembrane domain 6, reduces the hMCHR1 cell surface expression level to 20% of that observed for the wild-type receptor. Most of these mutant receptors are located intracellularly, as opposed to the wild-type receptor, which is located primarily on the cell surface. Immunoprecipitation experiments show that hMCHR1-T255A has reduced glycosylation compared with the wild-type receptor and is associated with the chaperone protein, calnexin, and it colocalizes in the endoplasmic reticulum with KDEL-containing proteins. We also demonstrate that a cell-permeable small molecule antagonist of hMCHR1 can function as a pharmacological chaperone to restore cell surface expression of this and other MCHR1 mutants to wild-type levels. Once rescued, the T255A mutant couples to Gq proteins as efficiently as the wild-type receptor. These data suggest that this single mutation produces an hMCHR1 that folds incorrectly, resulting in its retention in the endoplasmic reticulum, but once rescued to the cell surface can still function normally.

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