scholarly journals Cloning, Expression, and Functional Characterization of Relaxin Receptor (Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 7) Splice Variants from Human Fetal Membranes

Endocrinology ◽  
2007 ◽  
Vol 149 (3) ◽  
pp. 1277-1294 ◽  
Author(s):  
András Kern ◽  
Daniela Hubbard ◽  
Aaron Amano ◽  
Gillian D. Bryant-Greenwood
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
G Protein ◽  
Splice Variant ◽  
Splice Variants ◽  
Dominant Negative ◽  
Fetal Membranes ◽  
Leucine Rich Repeat ◽  
G Protein Coupled ◽  
Relaxin Receptor

The relaxin receptor [leucine-rich repeat-containing G protein-coupled receptor 7 (LGR7)] belongs to the leucine-rich repeat containing G protein-coupled receptors subgroup C. Three new LGR7 splice variants have been cloned from the human fetal membranes and shown to be truncated versions of the full-length receptor, encoded by different lengths of the extracellular domain. The expression of their mRNAs has been confirmed by both qualitative and quantitative PCR and shown to be higher in the chorion and decidua before, compared with after, spontaneous labor. When HEK293 cells were transfected with each LGR7 splice variant, their proteins were retained within the endoplasmic reticulum. However, the protein for the shortest variant was also secreted into the medium. We have characterized the intracellular functions and effects of these LGR7 variants on the function of the wild-type (WT)-LGR7. In coexpression studies, each splice variant interacted directly with the WT-LGR7 and exerted a dominant-negative effect on cAMP accumulation by the WT-LGR7 after relaxin treatment. This interaction resulted in the sequestration of the WT-LGR7 inside the cells by down-regulation of its maturation and cell surface delivery. The constitutive homodimerization of WT-LGR7 has been shown here to take place in the endoplasmic reticulum, and the presence of any one of the splice variants decreased this by the formation of heterodimers with the WT-LGR7, supporting the view that homodimerization is a prerequisite for receptor trafficking to the cell surface. These data suggest that the dominant-negative effects of the LGR7 splice variants expressed in the chorion and decidua could be functionally significant in the peripartal period by inhibiting the function of WT-LGR7 and dampening the responsiveness of these tissues to endogenous relaxin.

The Endoplasmic Reticulum Ca2+-pump SERCA2b Interacts with G Protein-coupled Receptors and Enhances their Expression at the Cell Surface

2007 ◽  
Vol 371 (3) ◽  
pp. 622-638 ◽  
Author(s):  
Jussi T. Tuusa ◽  
Piia M.H. Markkanen ◽  
Pirjo M. Apaja ◽  
Anna E. Hakalahti ◽  
Ulla E. Petäjä-Repo
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
G Protein Coupled

scholarly journals SAT-LB137 Rescue of Misfolded G-Protein Coupled Estrogen Receptor, GPER, from the Endoplasmic Reticulum via Natural and Synthetic Estrogens

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Milad Rouhimoghadam ◽  
Jing Dong ◽  
Peter Thomas ◽  
Edward Joseph Filardo
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
G Protein ◽  
Surface Expression ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
17Β Estradiol ◽  
G Protein Coupled

Abstract GPER bears structural and functional characteristics shared by members of the G-protein coupled receptor (GPCR) superfamily, the largest class of cell surface receptors, with more than 800 members encoded in the human genome. GPER is localized predominately in intracellular membranes, in many but not all cell types, and its surface expression is modulated by steroid hormones and during tissue homeostasis. An intracellular staining pattern is not unique among GPCRs, which deploy a diverse array of posttranslational regulatory mechanisms to determine cell surface expression, effectively regulating cognate ligand binding and activity. Here, we show nascent GPER undergoes strict quality control via endoplasmic reticulum associated degradation (ERAD) requiring direct poly-ubiquitinylation of GPER and valosin-containing protein VCP/p97-mediated segregation of misfolded proteins from the ER membrane to the cytoplasm for delivery to the 26S proteasome. Specifically, we find that inhibition of p97 using the pharmacological compound, CB-5083, or by doxycycline-inducible p97 shRNA results in the accumulation of immature glycosylated GPER in the ER. Inhibition of proteasome function facilitates anterograde trafficking with the transport of nonfunctional GPER to the plasma membrane as indicated by no increase in specific estrogen binding using 3H-17β-estradiol in a radioreceptor assay. The forward trafficking of misfolded GPER requires transit through the Golgi as treatment with brefeldin A (BFA) prevents GPER plasma membrane expression. Substitution of all three lysines (K333, K342, and K357) encoded in the cytoplasmic tail of GPER with arginines blunts its polyubiquitinylation and allows GPER to evade degradation by quality control but does not result in increased plasma membrane expression suggesting that additional structural motifs encoded within GPER control its anterograde trafficking. In contrast, functional GPER is recovered at the plasma membrane of human SKBR3 breast cancer cells treated with either 17β-estradiol or the GPER selective antagonist, G15, in the presence of cycloheximide resulting in increased surface GPER. Thus, our findings suggest that estrogens, both natural and synthetic, can function as pharmacochaperones capable of promoting the correct folding of GPER and enhanced expression of functional GPER at the plasma membrane.

scholarly journals Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

1998 ◽  
Vol 9 (4) ◽  
pp. 885-899 ◽  
Author(s):  
Christopher J. Stefan ◽  
Mark C. Overton ◽  
Kendall J. Blumer
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
G Protein ◽  
G Proteins ◽  
Structural Changes ◽  
G Protein Coupled Receptors ◽  
Wild Type ◽  
Proline Residue ◽  
G Protein Coupled ◽  
Constitutively Active

We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate agonist-induced structural changes leading to G protein activation. Constitutive signaling by mutant receptors is suppressed upon coexpression with wild-type, but not G protein coupling-defective, receptors. Wild-type receptors may therefore sequester a limiting pool of G proteins; this apparent “precoupling” of receptors and G proteins could facilitate signal production at sites where cell surface projections form during mating partner discrimination. Finally, rather than being expressed mainly at the cell surface, constitutively active pheromone receptors accumulate in post-endoplasmic reticulum compartments. This is in contrast to other defective membrane proteins, which apparently are targeted by default to the vacuole. We suggest that the quality-control mechanism that retains receptors in post-endoplasmic reticulum compartments may normally allow wild-type receptors to fold into their native, fully inactive conformations before reaching the cell surface. This may ensure that receptors do not trigger a response in the absence of agonist.

scholarly journals Understudied G Protein-Coupled Receptors in the Kidney

Nephron ◽  
2021 ◽  
pp. 1-4
Author(s):  
Nathan A. Zaidman ◽  
Jennifer L. Pluznick
Keyword(s):  
Renal Function ◽  
Cell Surface ◽  
Gene Family ◽  
G Protein ◽  
External Environment ◽  
G Protein Coupled Receptors ◽  
Surface Proteins ◽  
Cell Surface Proteins ◽  
Large Subset ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are cell surface proteins which play a key role in allowing cells, tissues, and organs to respond to changes in the external environment in order to maintain homeostasis. Despite the fact that GPCRs are known to play key roles in a variety of tissues, there are a large subset of GPCRs that remain poorly studied. In this minireview, we will summarize what is known regarding the “understudied” GPCRs with respect to renal function, and in so doing will highlight the promise represented by studying this gene family.

scholarly journals Human Homologs of the Putative G Protein-Coupled Membrane Progestin Receptors (mPRα, β, and γ) Localize to the Endoplasmic Reticulum and Are Not Activated by Progesterone

2006 ◽  
Vol 20 (12) ◽  
pp. 3146-3164 ◽  
Author(s):  
Tom Krietsch ◽  
Maria Sofia Fernandes ◽  
Jukka Kero ◽  
Ralf Lösel ◽  
Maria Heyens ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
G Protein ◽  
Cell Lines ◽  
Spotted Seatrout ◽  
Membrane Receptors ◽  
Takifugu Rubripes ◽  
Camp Production ◽  
Intact Cells ◽  
Progestin Receptors ◽  
G Protein Coupled

Abstract The steroid hormone progesterone exerts pleiotrophic functions in many cell types. Although progesterone controls transcriptional activation through binding to its nuclear receptors, it also initiates rapid nongenomic signaling events. Recently, three putative membrane progestin receptors (mPRα, β, and γ) with structural similarity to G protein-coupled receptors have been identified. These mPR isoforms are expressed in a tissue-specific manner and belong to the larger, highly conserved family of progestin and adiponectin receptors found in plants, eubacteria, and eukaryotes. The fish mPRα has been reported to mediate progesterone-dependent MAPK activation and inhibition of cAMP production through coupling to an inhibitory G protein. To functionally characterize the human homologs, we established human embryonic kidney 293 and MDA-MB-231 cell lines that stably express human mPRα, β, or γ. For comparison, we also established cell lines expressing the mPRα cloned from the spotted seatrout (Cynoscion nebulosus) and Japanese pufferfish (Takifugu rubripes). Surprisingly, we found no evidence that human or fish mPRs regulate cAMP production or MAPK (ERK1/2 or p38) activation upon progesterone stimulation. Furthermore, the mPRs did not couple to a highly promiscuous G protein subunit, Gαq5i, in transfection studies or provoke Ca2+ mobilization in response to progesterone. Finally, we demonstrate that transfected mPRs, as well as endogenous human mPRα, localize to the endoplasmic reticulum, and that their expression does not lead to increased progestin binding either in membrane preparations or in intact cells. Our results therefore do not support the concept that mPRs are plasma membrane receptors involved in transducing nongenomic progesterone actions.

scholarly journals Characterization of Two Fly LGR (Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor) Proteins Homologous to Vertebrate Glycoprotein Hormone Receptors: Constitutive Activation of Wild-Type Fly LGR1 But Not LGR2 in Transfected Mammalian Cells**This study was supported by NIH Grant HD-23273. The GenBank submission number for fly LGR2 is AF274591.

Endocrinology ◽  
2000 ◽  
Vol 141 (11) ◽  
pp. 4081-4090 ◽  
Author(s):  
Shinya Nishi ◽  
Sheau Yu Hsu ◽  
Karen Zell ◽  
Aaron J. W. Hsueh
Keyword(s):  
G Protein ◽  
Hormone Receptors ◽  
Coupling Mechanism ◽  
Leucine Rich Repeat ◽  
G Protein Coupled Receptor ◽  
Glycoprotein Hormone ◽  
Glycoprotein Hormone Receptors ◽  
Signaling Mechanisms ◽  
G Protein Coupled

Abstract The receptors for lutropin (LH), FSH, and TSH belong to the large G protein-coupled receptor (GPCR) superfamily and are unique in having a large N-terminal extracellular (ecto-) domain important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of a large family of the leucine-rich repeat-containing, G protein-coupled receptors (LGRs) with at least seven members in mammals. Based on the sequences of mammalian glycoprotein hormone receptors, we have identified a new LGR in Drosophila melanogaster and named it as fly LGR2 to distinguish it from the previously reported fly LH/FSH/TSH receptor (renamed as fly LGR1). Genomic analysis indicated the presence of 10 exons in fly LGR2 as compared with 16 exons in fly LGR1. The deduced fly LGR2 complementary DNA (cDNA) showed 43 and 64% similarity to the fly LGR1 in the ectodomain and transmembrane region, respectively. Comparison of 12 LGRs from diverse species indicated that these proteins can be divided into three subfamilies and fly LGR1 and LGR2 belong to different subfamilies. Potential signaling mechanisms were tested in human 293T cells overexpressing the fly receptors. Of interest, fly LGR1, but not LGR2, showed constitutive activity as reflected by elevated basal cAMP production in transfected cells. The basal activity of fly LGR1 was further augmented following point mutations of key residues in the intracellular loop 3 or transmembrane VI, similar to those found in patients with familial male precocious puberty. The present study reports the cloning of fly LGR2 and indicates that the G protein-coupling mechanism is conserved in fly LGR1 as compared with the mammalian glycoprotein hormone receptors. The characterization of fly receptors with features similar to mammalian glycoprotein hormone receptors allows a better understanding of the evolution of this unique group of GPCRs and future elucidation of their ligand signaling mechanisms.

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