Obesity-Linked Variants of Melanocortin-4 Receptor Are Misfolded in the Endoplasmic Reticulum and Can Be Rescued to the Cell Surface by a Chemical Chaperone

Abstract Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain where it controls food intake. Many obesity-linked MC4R variants are poorly expressed at the plasma membrane and are retained intracellularly. We have studied the intracellular localization of four obesity-linked MC4R variants, P78L, R165W, I316S, and I317T, in immortalized neurons. We find that these variants are all retained in the endoplasmic reticulum (ER), are ubiquitinated to a greater extent than the wild-type (wt) receptor, and induce ER stress with increased levels of ER chaperones as compared with wt-MC4R and appearance of CCAAT/enhancer-binding protein homologous protein. Expression of the X-box-binding-protein-1 with selective activation of a protective branch of the unfolded protein response did not have any effect on the cell surface expression of MC4R-I316S. Conversely, the pharmacological chaperone 4-phenyl butyric acid (PBA) increased the cell surface expression of wt-MC4R, MC4R-I316S, and I317T by more than 40%. PBA decreased ubiquitination of MC4R-I316S and prevented ER stress induced by expression of the mutant, suggesting that the drug functions to promote MC4R folding. MC4R-I316S rescued to the cell surface is functional, with a 52% increase in agonist-induced cAMP production, as compared with untreated cells. Also direct inhibition of wt-MC4R and MC4R-I316S ubiquitination by a specific inhibitor of the ubiquitin-activating enzyme 1 increased by approximately 40% the expression of the receptors at the cell surface, and the effects of PBA and ubiquitin-activating enzyme 1 were additive. These data offer a cell-based rationale that drugs that improve MC4R folding or decrease ER-associated degradation of the receptor may function to treat some forms of hereditary obesity.

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The immunogenicity of VP7, a rotavirus antigen resident in the endoplasmic reticulum, is enhanced by cell surface expression.

Journal of Virology ◽

10.1128/jvi.64.10.4776-4783.1990 ◽

1990 ◽

Vol 64 (10) ◽

pp. 4776-4783 ◽

Cited By ~ 24

Author(s):

M E Andrew ◽

D B Boyle ◽

P L Whitfeld ◽

L J Lockett ◽

I D Anthony ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cell Surface ◽

Surface Expression ◽

Cell Surface Expression

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Pleiotropic functions of the transmembrane domain 6 of human melanocortin-4 receptor

Journal of Molecular Endocrinology ◽

10.1530/jme-12-0161 ◽

2012 ◽

Vol 49 (3) ◽

pp. 237-248 ◽

Cited By ~ 21

Author(s):

Hui Huang ◽

Ya-Xiong Tao

Keyword(s):

Cell Surface ◽

Structure Function ◽

Transmembrane Domain ◽

Mapk Pathway ◽

Surface Expression ◽

Camp Signaling ◽

Cell Surface Expression ◽

Melanocortin 4 Receptor ◽

Function Relationship ◽

Relationship Of

The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis and has emerged as a premier target for obesity treatment. Numerous mutations in transmembrane domain 6 (TM6) of MC4R resulting in functional alterations have been identified in obese patients. Several mutagenesis studies also provided some data suggesting the importance of this domain in receptor function. To gain a better understanding of the structure–function relationship of the receptor, we performed alanine-scanning mutagenesis in TM6 to determine the functions of side chains. Of the 31 residues, two were important for cell surface expression, five were indispensable for α-melanocyte-stimulating hormone (α-MSH) and β-MSH binding, and six were important for signaling in the Gs–cAMP–PKA pathway. H264A, targeted normally to the plasma membrane, was undetectable by competitive binding assay and severely defective in basal and stimulated cAMP production and ERK1/2 phosphorylation. Nine mutants had decreased basal cAMP signaling. Seven mutants were constitutively active in cAMP signaling and their basal activities could be inhibited by two MC4R inverse agonists, Ipsen 5i and ML00253764. Five mutants were also constitutively active in the MAPK pathway with enhanced basal ERK1/2 phosphorylation. In summary, our study provided comprehensive data on the structure–function relationship of the TM6 of MC4R. We identified residues that are important for cell surface expression, ligand binding, cAMP generation, and residues for maintaining the WT receptor in active conformation. We also reported constitutive activation of the MAPK pathway and biased signaling. These data will be useful for rationally designing MC4R agonists and antagonists for treatment of eating disorders.

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Analysis of functional domains of the v-fms-encoded protein of Susan McDonough strain feline sarcoma virus by linker insertion mutagenesis.

Molecular and Cellular Biology ◽

10.1128/mcb.7.9.3287 ◽

1987 ◽

Vol 7 (9) ◽

pp. 3287-3296 ◽

Cited By ~ 12

Author(s):

S D Lyman ◽

L R Rohrschneider

Keyword(s):

Cell Surface ◽

Biological Effects ◽

Intracellular Localization ◽

Surface Expression ◽

Cell Surface Expression ◽

Insertion Mutagenesis ◽

Focus Formation ◽

External Domain ◽

Sarcoma Virus

The Susan McDonough strain of feline sarcoma virus contains an oncogene, v-fms, which is capable of transforming fibroblasts in vitro. The mature protein product of the v-fms gene (gp140fms) is found on the surface of transformed cells; this glycoprotein has external, transmembrane, and cytoplasmic domains. To assess the functional role of these domains in transformation, we constructed a series of nine linker insertion mutations throughout the v-fms gene by using a dodecameric BamHI linker. The biological effects of these mutations on the function and intracellular localization of v-fms-encoded proteins were determined by transfecting the mutated DNA into Rat-2 cells. Most of the mutations within the external domain of the v-fms-encoded protein eliminated focus formation on Rat-2 cells; three of these mutations interfered with the glycosylation of the v-fms protein and interfered with formation of the mature gp140fms. One mutation in the external domain led to cell surface expression of v-fms protein even in the absence of complete glycosylational processing. Cell surface expression of mutated v-fms protein is probably necessary, but is not sufficient, for cell transformation since mutant v-fms protein was found on the surface of several nontransformed cell lines. Mutations that were introduced within the external domain had little effect on in vitro kinase activity, whereas mutations within the cytoplasmic domain all had strong inhibitory effects on this activity.

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Blocking intracellular degradation of the erythropoietin and asialoglycoprotein receptors by calpain inhibitors does not result in the same increase in the levels of their membrane and secreted forms

Biochemical Journal ◽

10.1042/bj3130391 ◽

1996 ◽

Vol 313 (2) ◽

pp. 391-399 ◽

Cited By ~ 22

Author(s):

Drorit NEUMANN ◽

Ming YUK HUAM ◽

Harvey F. LODISH ◽

Gerardo Z. LEDERKREMER

Keyword(s):

Endoplasmic Reticulum ◽

Cell Surface ◽

Surface Expression ◽

Cell Surface Expression ◽

Membrane Glycoprotein ◽

3T3 Cells ◽

Proteolytic Fragment ◽

Intracellular Degradation ◽

Nih 3T3 ◽

Calpain Inhibitors

The erythropoietin receptor (EPO-R), a type 1 membrane glycoprotein, is degraded mainly in the lysosomes or endosomes, whereas the asialoglycoprotein receptor (ASGP-R) H2a subunit, a type 2 membrane glycoprotein, is degraded exclusively in the endoplasmic reticulum. The present study describes compounds that inhibit the intracellular degradation of these receptors in an efficient manner. However, the levels of cell-surface expression and secretion of their soluble exoplasmic domains were not enhanced to the same extent. The calpain inhibitors N-acetyl-leucyl-leucyl-norleucinal(ALLN) and N-acetyl-leucyl-leucyl-methional (ALLM) inhibited EPO-R degradation profoundly. After 3 h of chase using Ba/F3 cells and NIH 3T3 fibroblasts expressing the EPO-R, virtually all of the receptor molecules were degraded, whereas 80% of the pulse-labelled receptor remained intact in the presence of the inhibitor. EPO-R cell-surface expression was elevated 1.5-fold after 1 h of incubation with ALLN. In the absence of protein synthesis, ALLN caused the accumulation of non-degraded EPO-R molecules in endosomes and lysosomes, as determined by double immunofluorescence labelling of NIH 3T3 cells expressing EPO-Rs. In Ba/F3 cells expressing a soluble EPO-R, ALLN treatment increased secretion of the soluble exoplasmic domain of the EPO-R 2-5-fold. Similarly, in NIH 3T3 cells singly transfected with the ASGP-R H2a subunit cDNA, ALLN inhibited degradation of the ASGP-R H2a subunit precursor, as well as the degradation of the 35 kDa proteolytic fragment corresponding to the receptor ectodomain, by 3-6-fold. However, accumulation of secreted proteolytic fragment in the medium was augmented in the presence of ALLN by only 1.75-fold. In cells expressing the G78R mutant of the ASGP-R H2a subunit, which is not cleaved to the 35 kDa fragment [Yuk and Lodish (1993) J. Cell Biol. 123,1735-1749], degradation of the precursor was inhibited. Overall, our data suggest the involvement of cysteine proteinases located in the endoplasmic reticulum, as well as in post-Golgi compartments, in degradation of the EPO-R and the ASGP-R H2a subunit. The much lower effect of the inhibitory compounds on cell-surface and secreted forms of the EPO-R and ASGP-R H2a subunit illustrates the complexity and the tight regulation of the cellular localization and stability of membrane proteins.

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Modulation of Transporter Associated with Antigen Processing (TAP)-Mediated Peptide Import into the Endoplasmic Reticulum by Flavivirus Infection

Journal of Virology ◽

10.1128/jvi.75.12.5663-5671.2001 ◽

2001 ◽

Vol 75 (12) ◽

pp. 5663-5671 ◽

Cited By ~ 49

Author(s):

Frank Momburg ◽

Arno Müllbacher ◽

Mario Lobigs

Keyword(s):

Endoplasmic Reticulum ◽

Cell Surface ◽

Mhc Class I ◽

Antigen Processing ◽

Surface Expression ◽

Class I ◽

Peptide Transport ◽

Cell Surface Expression ◽

Flavivirus Infection ◽

Mhc Class I Molecules

ABSTRACT In contrast to many other viruses that escape the cellular immune response by downregulating major histocompatibility complex (MHC) class I molecules, flavivirus infection can upregulate their cell surface expression. Previously we have presented evidence that during flavivirus infection, peptide supply to the endoplasmic reticulum is increased (A. Müllbacher and M. Lobigs, Immunity 3:207–214, 1995). Here we show that during the early phase of infection with different flaviviruses, the transport activity of the peptide transporter associated with antigen processing (TAP) is augmented by up to 50%. TAP expression is unaltered during infection, and viral but not host macromolecular synthesis is required for enhanced peptide transport. This study is the first demonstration of transient enhancement of TAP-dependent peptide import into the lumen of the endoplasmic reticulum as a consequence of a viral infection. We suggest that the increased supply of peptides for assembly with MHC class I molecules in flavivirus-infected cells accounts for the upregulation of MHC class I cell surface expression with the biological consequence of viral evasion of natural killer cell recognition.

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