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

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.

Download Full-text

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

Molecular Biology of the Cell ◽

10.1091/mbc.e07-09-0942 ◽

2008 ◽

Vol 19 (4) ◽

pp. 1540-1547 ◽

Cited By ~ 222

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.

Download Full-text

Expression of the Third Intracellular Loop of the δ-Opioid Receptor Inhibits Signaling by Opioid Receptors and Other G Protein-Coupled Receptors

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.105.089946 ◽

2005 ◽

Vol 315 (3) ◽

pp. 1368-1379 ◽

Cited By ~ 23

Author(s):

Evangelia Morou ◽

Zafiroula Georgoussi

Keyword(s):

G Protein ◽

Opioid Receptor ◽

Opioid Receptors ◽

G Protein Coupled Receptors ◽

Intracellular Loop ◽

The Third ◽

Third Intracellular Loop ◽

Δ Opioid Receptor ◽

G Protein Coupled

Download Full-text

The Third Intracellular Loop of the Human Somatostatin Receptor 5 Is Crucial for Arrestin Binding and Receptor Internalization after Somatostatin Stimulation

Molecular Endocrinology ◽

10.1210/me.2007-0068 ◽

2008 ◽

Vol 22 (3) ◽

pp. 676-688 ◽

Cited By ~ 28

Author(s):

Erika Peverelli ◽

Giovanna Mantovani ◽

Davide Calebiro ◽

Andrea Doni ◽

Sara Bondioni ◽

...

Keyword(s):

Somatostatin Receptor ◽

Hormone Secretion ◽

Serine Phosphorylation ◽

Receptor Internalization ◽

Receptor Interaction ◽

Wild Type ◽

Intracellular Loop ◽

Structural Domains ◽

The Third ◽

Third Intracellular Loop

Abstract Somatostatin (SS) is a widely distributed polypeptide that exerts inhibitory effects on hormone secretion and cell proliferation by interacting with five different receptors (SST1-SST5). β-Arrestins have been implicated in regulating SST internalization, but the structural domains mediating this effect are largely unknown. The aim of this study was to characterize the intracellular mechanisms responsible for internalization of human SST5 in the rat pituitary cell line GH3 and to identify the SST5 structural domains involved in this process. To this purpose we evaluated, by fluorescence microscopy and biochemical assay, the ability of wild-type, progressive C-terminal truncated and third cytoplasmatic loop mutants SST5-DsRed to associate with β-arrestin-enhanced green fluorescent protein and to internalize under SS28 stimulation. The truncated mutants were comparable to the wild-type receptor with respect to recruitment of β-arrestin-2 and internalization, whereas the third loop mutants R240W, S242A, and T247A showed the abolishment or reduction of arrestin association and a significant reduction of receptor internalization (14.4%, 29%, and 30.9% vs. 52.4% of wild type) and serine phosphorylation upon SS28 stimulation. Moreover, we evaluated the ability of simultaneous mutation of these three residues (R240, S242, and T247) and C-terminal truncated receptors to internalize. The progressive truncation of the C-terminal tail resulted in a progressive increased internalization (21.6%, 36.7%, and 41%, respectively) with respect to the full-length total third-loop mutant (15%). In conclusion, our results indicate the SST5 third intracellular loop as an important mediator of β-arrestin/receptor interaction and receptor internalization, whereas they suggest that residues 328–347 within the C terminus may play an inhibitory role in receptor internalization.

Download Full-text

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

Life Science Alliance ◽

10.26508/lsa.202000746 ◽

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.

Download Full-text

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

Journal of Cell Science ◽

10.1242/jcs.115.1.165 ◽

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.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj20150876 ◽

2015 ◽

Vol 472 (3) ◽

pp. 287-295 ◽

Cited By ~ 13

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.

Download Full-text

Characterization of Intracellular Signaling Mediated by Human Somatostatin Receptor 5: Role of the DRY Motif and the Third Intracellular Loop

Endocrinology ◽

10.1210/en.2008-1785 ◽

2009 ◽

Vol 150 (7) ◽

pp. 3169-3176 ◽

Cited By ~ 19

Author(s):

Erika Peverelli ◽

Andrea G. Lania ◽

Giovanna Mantovani ◽

Paolo Beck-Peccoz ◽

Anna Spada

Keyword(s):

Cell Proliferation ◽

Intracellular Calcium ◽

Intracellular Signaling ◽

Somatostatin Receptor ◽

Intracellular Camp ◽

Wild Type ◽

Intracellular Loop ◽

The Third ◽

Dry Motif ◽

Third Intracellular Loop

Somatostatin (SST) exerts inhibitory effects on hormone secretion and cell proliferation by interacting with five different receptors (SST1-SST5) linked to multiple cellular effectors. The receptor structural domains involved in these effects have been only partially elucidated. The aim of the study was to investigate the molecular determinants mediating the interaction of the human SST5 with intracellular signaling in the pituitary cell line GH3, focusing on the BBXXB domain in the third intracellular loop and the DRY motif in the second intracellular loop. We analyzed the effects of the SST5 agonist BIM23206 on cAMP accumulation, intracellular calcium, GH secretion, cell proliferation, and ERK1/2 phosphorylation in cells expressing either wild-type SST5 or mutant receptors, in particular the naturally occurring mutant R240W in the BBXXB domain and the D136A and R137A mutants in the DRY motif. We found that residues D136 and R137 were critical for SST5 signaling because their substitutions abolished all the intracellular responses. Conversely, third intracellular loop mutations resulted in receptor that inhibited intracellular cAMP levels similar to the wild-type (50 ± 9 vs. 53 ± 12% inhibition) but failed to mediate the other responses elicited by wild-type SST5, i.e. reduction of intracellular calcium levels as well as inhibition of ERK1/2. These events resulted in an absent inhibition of GH release and an impaired reduction of cell proliferation (38 ± 7 vs. 76 ± 6% inhibition in wild type, P < 0.05). These data indicate that different regions of SST5 are required for the activation of different signaling pathways.

Download Full-text