scholarly journals Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Protein-coupled Receptors

2008 ◽  
Vol 19 (4) ◽  
pp. 1540-1547 ◽  
Author(s):  
Nicolas F. Berbari ◽  
Andrew D. Johnson ◽  
Jacqueline S. Lewis ◽  
Candice C. Askwith ◽  
Kirk Mykytyn
Keyword(s):  
G Protein ◽  
Serotonin Receptor ◽  
Primary Cilia ◽  
Consensus Sequence ◽  
G Protein Coupled Receptors ◽  
Intracellular Loop ◽  
The Third ◽  
Third Intracellular Loop ◽  
G Protein Coupled ◽  
Ciliary Localization

Primary cilia are sensory organelles present on most mammalian cells. The functions of cilia are defined by the signaling proteins localized to the ciliary membrane. Certain G protein–coupled receptors (GPCRs), including somatostatin receptor 3 (Sstr3) and serotonin receptor 6 (Htr6), localize to cilia. As Sstr3 and Htr6 are the only somatostatin and serotonin receptor subtypes that localize to cilia, we hypothesized they contain ciliary localization sequences. To test this hypothesis we expressed chimeric receptors containing fragments of Sstr3 and Htr6 in the nonciliary receptors Sstr5 and Htr7, respectively, in ciliated cells. We found the third intracellular loop of Sstr3 or Htr6 is sufficient for ciliary localization. Comparison of these loops revealed a loose consensus sequence. To determine whether this consensus sequence predicts ciliary localization of other GPCRs, we compared it with the third intracellular loop of all human GPCRs. We identified the consensus sequence in melanin-concentrating hormone receptor 1 (Mchr1) and confirmed Mchr1 localizes to primary cilia in vitro and in vivo. Thus, we have identified a putative GPCR ciliary localization sequence and used this sequence to identify a novel ciliary GPCR. As Mchr1 mediates feeding behavior and metabolism, our results implicate ciliary signaling in the regulation of body weight.

Spinophilin regulates Ca2+ signalling by binding the N-terminal domain of RGS2 and the third intracellular loop of G-protein-coupled receptors

2005 ◽  
Vol 7 (4) ◽  
pp. 405-411 ◽  
Author(s):  
Xinhua Wang ◽  
Weizhong Zeng ◽  
Abigail A. Soyombo ◽  
Wei Tang ◽  
Elliott M. Ross ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Intracellular Loop ◽  
The Third ◽  
Terminal Domain ◽  
Third Intracellular Loop ◽  
G Protein Coupled

scholarly journals HTR6 and SSTR3 ciliary targeting relies on both IC3 loops and C-terminal tails

2020 ◽  
Vol 4 (3) ◽  
pp. e202000746
Author(s):  
Pablo Barbeito ◽  
Yuki Tachibana ◽  
Raquel Martin-Morales ◽  
Paula Moreno ◽  
Kirk Mykytyn ◽  
...  
Keyword(s):  
Serotonin Receptor ◽  
Primary Cilia ◽  
Molecular Mechanisms ◽  
Somatostatin Receptor ◽  
G Protein Coupled Receptors ◽  
The Third ◽  
Membrane Protrusions ◽  
Intracellular Loops ◽  
G Protein Coupled ◽  
Shed Light

G protein-coupled receptors (GPCRs) are the most common pharmacological target in human clinical practice. To perform their functions, many GPCRs must accumulate inside primary cilia, microtubule-based plasma membrane protrusions working as cellular antennae. Nevertheless, the molecular mechanisms underlying GPCR ciliary targeting remain poorly understood. Serotonin receptor 6 (HTR6) and somatostatin receptor 3 (SSTR3) are two brain-enriched ciliary GPCRs involved in cognition and pathologies such as Alzheimer’s disease and cancer. Although the third intracellular loops (IC3) of HTR6 and SSTR3 suffice to target non-ciliary GPCRs to cilia, these IC3s are dispensable for ciliary targeting of HTR6 and SSTR3 themselves, suggesting these GPCRs contain additional ciliary targeting sequences (CTSs). Herein, we discover and characterize novel CTSs in HTR6 and SSTR3 C-terminal tails (CT). These CT-CTSs (CTS2) act redundantly with IC3-CTSs (CTS1), each being sufficient for ciliary targeting. In HTR6, RKQ and LPG motifs are critical for CTS1 and CTS2 function, respectively, whereas in SSTR3 these roles are mostly fulfilled by AP[AS]CQ motifs in IC3 and juxtamembrane residues in CT. Furthermore, we shed light on how these CTSs promote ciliary targeting by modulating binding to ciliary trafficking adapters TULP3 and RABL2.

scholarly journals Recycling ability of the mouse and the human neurotensin type 2 receptors depends on a single tyrosine residue

2002 ◽  
Vol 115 (1) ◽  
pp. 165-173
Author(s):  
Stéphane Martin ◽  
Jean-Pierre Vincent ◽  
Jean Mazella
Keyword(s):  
G Protein ◽  
Kinase Inhibitor ◽  
Site Directed Mutagenesis ◽  
Tyrosine Residue ◽  
Receptor Recycling ◽  
Intracellular Loop ◽  
Structural Element ◽  
The Third ◽  
Third Intracellular Loop ◽  
G Protein Coupled

Receptor recycling plays a key role in the modulation of cellular responses to extracellular signals. The purpose of this work was to identify residues in G-protein coupled neurotensin receptors that are directly involved in recycling. Both the high affinity receptor-1 (NTR1) and the levocabastine-sensitive NTR2 are internalized after neurotensin binding. Here, we show that only the mouse NTR2 recycled to the plasma membrane, whereas the rat NTR1 and the human NTR2 did not. Using site-directed mutagenesis, we demonstrate that tyrosine 237 in the third intracellular loop is crucial for recycling of the mouse NTR2. We show that the mouse NTR2 is phosphorylated on tyrosine residues by NT. This phosphorylation is essential for receptor recycling since the tyrosine kinase inhibitor genistein blocks this process. The absence of recycling observed with the human NTR2 could be completely explained by the presence of a cysteine instead of a tyrosine in position 237. Indeed, substitution of this cysteine by a tyrosine gave a mutant receptor that has acquired the ability to recycle to the cell surface after neurotensin-induced internalization. This work demonstrates that a single tyrosine residue in the third intracellular loop of a G-protein-coupled receptor is responsible for receptor phosphorylation and represents an essential structural element for receptor recycling.

scholarly journals Down-regulation of PAR1 activity with a pHLIP-based allosteric antagonist induces cancer cell death

2015 ◽  
Vol 472 (3) ◽  
pp. 287-295 ◽  
Author(s):  
Kelly E. Burns ◽  
Damien Thévenin
Keyword(s):  
Cell Death ◽  
Cancer Cells ◽  
G Protein ◽  
Cancer Cell ◽  
Intracellular Loop ◽  
The Third ◽  
Cancer Cell Death ◽  
Third Intracellular Loop ◽  
Ph Dependent ◽  
G Protein Coupled

A pH(Low) Insertion Peptide (pHLIP)-based construct derived from the third intracellular loop (i3) of a G protein-coupled receptor (GPCR) induces a concentration- and pH-dependent cytotoxicity in cancer cells by down-regulating receptor activity. This strategy allows for a more selective intracellular delivery than current approaches.

scholarly journals HTR6 and SSTR3 targeting to primary cilia

2021 ◽  
Author(s):  
Pablo Barbeito ◽  
Francesc R. Garcia-Gonzalo
Keyword(s):  
Serotonin Receptor ◽  
Primary Cilia ◽  
Somatostatin Receptor ◽  
G Protein Coupled Receptors ◽  
Signal Transducers ◽  
Neuronal Cilia ◽  
Intracellular Loops ◽  
Oncologic Diseases ◽  
G Protein Coupled ◽  
Ciliary Localization

Primary cilia are hair-like projections of the cell membrane supported by an inner microtubule scaffold, the axoneme, which polymerizes out of a membrane-docked centriole at the ciliary base. By working as specialized signaling compartments, primary cilia provide an optimal environment for many G protein-coupled receptors (GPCRs) and their effectors to efficiently transmit their signals to the rest of the cell. For this to occur, however, all necessary receptors and signal transducers must first accumulate at the ciliary membrane. Serotonin receptor 6 (HTR6) and Somatostatin receptor 3 (SSTR3) are two GPCRs whose signaling in brain neuronal cilia affects cognition and is implicated in psychiatric, neurodegenerative, and oncologic diseases. Over a decade ago, the third intracellular loops (IC3s) of HTR6 and SSTR3 were shown to contain ciliary localization sequences (CLSs) that, when grafted onto non-ciliary GPCRs, could drive their ciliary accumulation. Nevertheless, these CLSs were dispensable for ciliary targeting of HTR6 and SSTR3, suggesting the presence of additional CLSs, which we have recently identified in their C-terminal tails. Herein, we review the discovery and mapping of these CLSs, as well as the state of the art regarding how these CLSs may orchestrate ciliary accumulation of these GPCRs by controlling when and where they interact with the ciliary entry and exit machinery via adaptors such as TULP3, RABL2 and the BBSome.

scholarly journals Untangling ciliary access and enrichment of two rhodopsin-like receptors using quantitative fluorescence microscopy reveals cell-specific sorting pathways

2017 ◽  
Vol 28 (4) ◽  
pp. 554-566 ◽  
Author(s):  
Ivayla I. Geneva ◽  
Han Yen Tan ◽  
Peter D. Calvert
Keyword(s):  
Primary Cilia ◽  
Somatostatin Receptor ◽  
Optical Systems ◽  
Rod Photoreceptor ◽  
Intracellular Loop ◽  
The Third ◽  
Cell Compartments ◽  
Third Intracellular Loop ◽  
Apical Membranes ◽  
G Protein Coupled

Resolution limitations of optical systems are major obstacles for determining whether proteins are enriched within cell compartments. Here we use an approach to determine the degree of membrane protein ciliary enrichment that quantitatively accounts for the differences in sampling of the ciliary and apical membranes inherent to confocal microscopes. Theory shows that cilia will appear more than threefold brighter than the surrounding apical membrane when the densities of fluorescently labeled proteins are the same, thus providing a benchmark for ciliary enrichment. Using this benchmark, we examined the ciliary enrichment signals of two G protein–coupled receptors (GPCRs)—the somatostatin receptor 3 and rhodopsin. Remarkably, we found that the C-terminal VxPx motif, required for efficient enrichment of rhodopsin within rod photoreceptor sensory cilia, inhibited enrichment of the somatostatin receptor in primary cilia. Similarly, VxPx inhibited primary cilium enrichment of a chimera of rhodopsin and somatostatin receptor 3, where the dual Ax(S/A)xQ ciliary targeting motifs within the third intracellular loop of the somatostatin receptor replaced the third intracellular loop of rhodopsin. Rhodopsin was depleted from primary cilia but gained access, without being enriched, with the dual Ax(S/A)xQ motifs. Ciliary enrichment of these GPCRs thus operates via distinct mechanisms in different cells.

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