scholarly journals Loss-of-Function Mutations in the Human Luteinizing Hormone Receptor Predominantly Cause Intracellular Retention

Endocrinology ◽  
2016 ◽  
Vol 157 (11) ◽  
pp. 4364-4377 ◽  
Author(s):  
Claire Louise Newton ◽  
Ross Calley Anderson ◽  
Arieh Anthony Katz ◽  
Robert Peter Millar
Keyword(s):  
Luteinizing Hormone ◽  
Cell Surface ◽  
Ligand Binding ◽  
Hormone Receptors ◽  
Receptor Activation ◽  
Luteinizing Hormone Receptor ◽  
Cell Surface Expression ◽  
Receptor Function ◽  
Loss Of Function ◽  
Pharmacological Chaperone

Mutations in G protein–coupled receptors (GPCRs) have been identified for many endocrine hormone signaling deficiencies. Inactivating mutations can impair ligand binding, receptor activation/coupling to signaling pathways, or can cause receptor misfolding and consequent impaired expression at the cell membrane. Here we examine the cell surface expression, ligand binding, and signaling of a range of mutant human luteinizing hormone receptors (LHRs) identified as causing reproductive dysfunction in human patients. The data obtained reveal how mutations in GPCRs can have diverse and severely deleterious effects on receptor function. Furthermore, it was found that impaired functionality of the majority of the mutant LHRs was due to reduced expression at the cell surface (14/20) while only two mutations caused impaired binding affinity and two impaired in signaling. An additional two mutations were found to cause no impairment of receptor function. These data demonstrate that the majority of LHR mutations lead to intracellular retention and highlight the potential for novel pharmacological chaperone therapeutics that can “rescue” expression/function of retained mutant GPCRs.

scholarly journals Functional Characterization of Naturally Occurring Pathogenic Mutations in the Human Leptin Receptor

Endocrinology ◽  
2008 ◽  
Vol 149 (12) ◽  
pp. 6043-6052 ◽  
Author(s):  
Wendy Kimber ◽  
Frank Peelman ◽  
Xavier Prieur ◽  
Teresia Wangensteen ◽  
Stephen O'Rahilly ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Binding Site ◽  
Molecular Mechanisms ◽  
Leptin Receptor ◽  
Receptor Activation ◽  
Cell Surface Expression ◽  
Missense Mutations ◽  
Naturally Occurring

We have recently reported the first naturally occurring missense mutations in the leptin receptor (LR) in patients with severe obesity. We have examined the molecular mechanisms by which these extracellular domain mutations disrupt LR signaling. The Ala409Glu mutant receptor is expressed at the cell surface, binds leptin normally but fails to signal to downstream pathways. A409 is present on the surface-exposed region of the Ig-like domain that forms the binding site III for interaction with leptin. This binding site does not appear to contribute to the binding affinity of leptin to its receptor but is critical for receptor activation in response to ligand binding. The Trp664Arg and His684Pro mutations are predicted to impair receptor folding. Both mutants result in a complete inability to signal to downstream pathways despite evidence for some residual cell surface expression and ligand binding. The Arg612His mutant falls in the second subdomain of the high-affinity binding site for leptin, and results in a receptor that shows evidence for intracellular retention but retains some residual signaling. These studies, which represent the first detailed characterization of the functional properties of naturally occurring missense mutations in the human LR, indicate that most such mutations affect receptor folding and expression at the cell surface rather than primarily impairing ligand binding. The exception is Ala409Glu, which interferes with the coupling of ligand binding to receptor activation. Naturally occurring mutations associated with human obesity are valuable tools with which to explore structure/function relationships within the LR.

scholarly journals Functional Characterization and Structural Modeling of Obesity Associated Mutations in the Melanocortin 4 Receptor

Endocrinology ◽  
2008 ◽  
Vol 150 (1) ◽  
pp. 114-125 ◽  
Author(s):  
Karen Tan ◽  
Irina D. Pogozheva ◽  
Giles S. H. Yeo ◽  
Dirk Hadaschik ◽  
Julia M. Keogh ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Functional Characterization ◽  
Structural Modeling ◽  
Receptor Activation ◽  
Cell Surface Expression ◽  
Structural Basis ◽  
Conformational Rearrangement ◽  
Mc4r Gene ◽  
Melanocortin 4 Receptor

Mutations in the melanocortin 4 receptor (MC4R) gene are the most common known cause of monogenic human obesity. The MC4R gene was sequenced in 2000 subjects with severe early-onset obesity. We detected seven different nonsense and 19 nonsynonymous mutations in a total of 94 probands, some of which have been reported previously by others. We functionally characterized the 11 novel obesity associated missense mutations. Seven of these mutants (L54P, E61K, I69T, S136P, M161T, T162I, and I269N) showed impaired cell surface trafficking, reduced level of maximal binding of the radioligand [125I]NDP-MSH, and reduced ability to generate cAMP in response to ligand. Four mutant MC4Rs (G55V, G55D, S136F, and A303T) displayed cell surface expression and agonist binding similar to the wild-type receptor but showed impaired cAMP production, suggesting that these residues are likely to be critical for conformational rearrangement essential for receptor activation. Homology modeling of these mutants using a model of MC4R based on the crystal structure of the β2-adrenoreceptor was used to provide insights into the possible structural basis for receptor dysfunction. Transmembrane (TM) domains 1, 3, 6, 7, and peripheral helix 8 appear to participate in the agonist-induced conformational rearrangement necessary for coupling of ligand binding to signaling. We conclude that G55V, G55D, S136F, and A303T mutations are likely to strengthen helix-helix interactions between TM1 and TM2, TM3 and TM6, and TM7 and helix 8, respectively, preventing relative movement of these helices during receptor activation. The combination of functional studies and structural modeling of naturally occurring pathogenic mutations in MC4R can provide valuable information regarding the molecular mechanism of MC4R activation and its dysfunction in human disease. Among obesity-associated melanocortin-4 receptor mutations, four transmembrane domains and peripheral helix 8 are necessary for coupling of ligand binding to signaling.

Rescue of cell surface expression and signalling of mutant follicle-stimulating hormone receptors

Endocrinology ◽  
2021 ◽  
Author(s):  
Sharika Hanyroup ◽  
Ross C Anderson ◽  
Selvaraj Nataraja ◽  
Henry N Yu ◽  
Robert P Millar ◽  
...  
Keyword(s):  
Cell Surface ◽  
Hormone Receptors ◽  
Follicle Stimulating Hormone ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Pharmacological Chaperone ◽  
Pharmacological Chaperones ◽  
And Function ◽  
G Protein Coupled ◽  
Stimulating Hormone

Abstract Mutations in G protein-coupled receptors (GPCRs) underlie numerous diseases. Many cause receptor misfolding and failure to reach the cell surface. Pharmacological chaperones are cell-permeant small-molecules that engage nascent mutant GPCRs in the endoplasmic reticulum, stabilising folding and ‘rescuing’ cell surface expression. We previously demonstrated rescue of cell surface expression of luteinising hormone receptor mutants by an allosteric agonist. Here we demonstrate that a similar approach can be employed to rescue mutant follicle-stimulating hormone receptors (FSHRs) with poor cell surface expression using a small-molecule FSHR agonist, CAN1404. Seventeen FSHR mutations described in patients with reproductive dysfunction were expressed in HEK 293T cells and cell surface expression was determined by ELISA of epitope-tagged FSHRs before/after treatment with CAN1404. Cell surface expression was severely reduced to ≤18% of wild-type (WT) for eleven, modestly reduced to 66–84% of WT for four and was not reduced for two. Of the eleven with severely reduced cell surface expression, restoration to ≥57% of WT levels was achieved for six by treatment with 1 µM CAN1404 for 24 h and a corresponding increase in FSH-induced signalling was observed for four of these, indicating restored functionality. Therefore, CAN1404 acts as a pharmacological chaperone and can rescue cell surface expression and function of certain mutant FSHRs with severely reduced cell surface expression. These findings aid in advancing the understanding of the effects of genetic mutations on GPCR function and provide a proof of therapeutic principle for FSHR PCs.

scholarly journals Differential Expression and Functional Characterization of Luteinizing Hormone Receptor Splice Variants in Human Luteal Cells: Implications for Luteolysis

Endocrinology ◽  
2009 ◽  
Vol 150 (6) ◽  
pp. 2873-2881 ◽  
Author(s):  
Rachel E. Dickinson ◽  
Alan J. Stewart ◽  
Michelle Myers ◽  
Robert P. Millar ◽  
W. Colin Duncan
Keyword(s):  
Cell Surface ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Splice Variants ◽  
Primary Cultures ◽  
Surface Expression ◽  
Full Length ◽  
Luteinizing Hormone Receptor ◽  
Cell Surface Expression ◽  
Truncated Form

The human LH receptor (LHR) plays a key role in luteal function and the establishment of pregnancy through its interaction with the gonadotropins LH and human chorionic gonadotropin. We previously identified four splice variants of the LHR in human luteinized granulosa cells (LGCs) and corpora lutea (CL). Real-time quantitative PCR revealed that expression of the full-length LHR (LHRa) and the most truncated form (LHRd) changed significantly in CL harvested at different stages of the ovarian cycle (P < 0.01, ANOVA). LHRa expression was reduced in the late luteal CL (P < 0.05). Conversely, an increase in LHRd expression was observed in the late luteal CL (P < 0.01). Chronic manipulation of human chorionic gonadotropin in LGC primary cultures supported the in vivo findings. LHRd encodes a protein lacking the transmembrane and carboxyl terminal domains. COS-7 cells expressing LHRd were unable to produce cAMP in response to LH stimulation. COS-7 cells coexpressing LHRd and LHRa also failed to generate cAMP in response to LH, suggesting that this truncated form has a negative effect on the signaling of LHRa. Immunofluorescence staining of LGC and COS-7 cells implied that there is a reduction in cell surface expression of LHRa when LHRd is present. Overall, these results imply expression of LHR splice variants is regulated in the human CL. Furthermore, during functional luteolysis a truncated variant could modulate the cell surface expression and activity of full-length LHR.

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