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

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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.

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Activation of constitutive 5-hydroxytryptamine1B receptor by a series of mutations in the BBXXB motif: positioning of the third intracellular loop distal junction and its Goα protein interactions

Biochemical Journal ◽

10.1042/bj3430435 ◽

1999 ◽

Vol 343 (2) ◽

pp. 435-442 ◽

Cited By ~ 20

Author(s):

Petrus J. PAUWELS ◽

Agnès GOUBLE ◽

Thierry WURCH

Keyword(s):

Amino Acid ◽

Pertussis Toxin ◽

Receptor Activation ◽

Maximal Response ◽

Amino Acid Substitutions ◽

Wild Type ◽

Intracellular Loop ◽

Basic Residue ◽

The Third ◽

Third Intracellular Loop

Constitutive activity of the recombinant human 5-hydroxytryptamine1B (5-HT1B) receptor (RC code 2.1.5HT.01.B) was investigated by mutagenesis of the BBXXB motif (in which B represents a basic residue and X a non-basic residue) located in the C-terminal portion of the third intracellular loop. In contrast with wild-type 5-HT1B receptors, three receptor mutants (Thr313 → Lys, Thr313 → Arg and Thr313 → Gln) increased their agonist-independent guanosine 5′-[γ-[35S]thio]triphosphate binding response by 26-41%. This activity represented approx. 30% of the maximal response induced by 5-HT and could be reversed by the inverse agonists methiothepin and 3-(3-dimethylaminopropyl)-4-hydroxy-N-(4-pyridin-4-yl phenyl)-benzenamide (GR 55562). Enhanced agonist-independent and agonist-dependent 5-HT1B receptor activation was provided by co-expression of a pertussis toxin-resistant rat Goα Cys351 → Ile protein. The wild-type 5-HT1B receptor displayed a doubling in basal activity, whereas a spectrum of enhanced basal activities (313-571%) was observed with a series of diverse amino acid substitutions (isoleucine, glycine, asparagine, alanine, lysine, phenylalanine, glutamine and arginine) at the 5-HT1B receptor position 313 in the presence of pertussis toxin (100 ng/ml). Consequently, the constitutive 5-HT1B receptor activity can be modulated by the mutation of Thr313, and displays a graded range between 11% and 59% of maximal 5-HT1B receptor activation by 5-HT. No clear pattern is apparent in the framework of traditionally cited amino acid characteristics (i.e. residue size, charge or hydrophobicity) to explain the observed constitutive activities. The various amino acid substitutions that yielded enhanced activity are unlikely to make similar intramolecular interactions within the 5-HT1B receptor. It is hypothesized that the positioning of the junction between the third intracellular loop and transmembrane domain VI is altered by mutation of Thr313 in the BBXXB motif and thereby unmasks Gα-protein interaction points.

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The role of the third intracellular loop of the neutrophil N-formyl peptide receptor in G protein coupling

Biochemical Journal ◽

10.1042/bj2940581 ◽

1993 ◽

Vol 294 (2) ◽

pp. 581-587 ◽

Cited By ~ 36

Author(s):

E R Prossnitz ◽

O Quehenberger ◽

C G Cochrane ◽

R D Ye

Keyword(s):

Amino Acids ◽

G Protein ◽

Formyl Peptide Receptor ◽

Wild Type ◽

Intracellular Loop ◽

G Protein Coupling ◽

Protein Coupling ◽

The Third ◽

Third Intracellular Loop ◽

Formyl Peptide

The G-protein-coupled N-formyl peptide receptor (FPR) contains one of the smallest known third intracellular loops of this class of receptors, consisting of only 15 amino acids. To study the role of this region of the receptor in G protein coupling and signal transduction, we generated a deletion mutant (D3i) in which 10 amino acids of the loop were removed, as well as a series of site-directed mutants containing substitutions of the charged and polar amino acids of this loop. The D3i mutant, expressed at normal levels on the cell surface, displayed a KD for labelled N-formyl-Met-Leu-Phe ([3H]FMLP) of 165 nM. This value compares with a KD for the wild-type FPR of 1.0 nM, or 20 nM in the presence of guanosine 5′-[gamma-thio]triphosphate, which uncouples G proteins from the receptor. These results indicate that D3i contains significant structural defects, beyond the disruption of G protein coupling, that affect ligand binding properties. Ten site-directed mutants generated in the third intracellular loop (T226A, K227E, H229A, K230Q, K235Q, S236A, S236A/S237G, R238G, R241E and S244A) displayed KD values between 0.5 and 1.0 nM, with expression levels between 22% (K227E) and 111% (H229A) of that of wild type receptor. The capacity of the mutants for signal transductions was determined by measuring intracellular Ca2+ mobilization. Eight of the ten mutants displayed EC50 values for FMLP of between 0.07 and 0.9 nM, as compared with 0.12 nM for the wild-type receptor. The two mutants K227E and R238G had EC50 values of 2.7 and 2.9 nM respectively. The increase in EC50 could be accounted for partially by the low levels of receptor expression. All ten mutants gave maximum levels of Ca2+ mobilization similar to that produced by the wild-type FPR. These results contradict the conclusions reached with other G-protein-coupled receptors and indicate that the third intracellular loop of the FPR does not have a critical role in the functional coupling of G proteins.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e16-07-0549 ◽

2017 ◽

Vol 28 (4) ◽

pp. 554-566 ◽

Cited By ~ 8

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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Differential Intracellular Signaling through PAC1 Isoforms as a Result of Alternative Splicing in the First Extracellular Domain and the Third Intracellular Loop

Molecular Pharmacology ◽

10.1124/mol.107.035477 ◽

2007 ◽

Vol 72 (1) ◽

pp. 103-111 ◽

Cited By ~ 30

Author(s):

Mina Ushiyama ◽

Ryuji Ikeda ◽

Hideki Sugawara ◽

Morikatsu Yoshida ◽

Kenji Mori ◽

...

Keyword(s):

Alternative Splicing ◽

Intracellular Signaling ◽

Extracellular Domain ◽

Intracellular Loop ◽

The Third ◽

Third Intracellular Loop

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Cross-talk between m1 muscarinic acetylcholine and beta 2-adrenergic receptors. cAMP and the third intracellular loop of m1 muscarinic receptors confer heterologous regulation.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)53050-7 ◽

1993 ◽

Vol 268 (11) ◽

pp. 7949-7957

Author(s):

N.H. Lee ◽

C.M. Fraser

Keyword(s):

Muscarinic Receptors ◽

Cross Talk ◽

Adrenergic Receptors ◽

Intracellular Loop ◽

Muscarinic Acetylcholine ◽

The Third ◽

Third Intracellular Loop ◽

Beta 2 ◽

M1 Muscarinic ◽

M1 Muscarinic Receptors

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Octreotide and Pasireotide Combination Treatment in Somatotroph Tumor Cells: Predominant Role of SST2 in Mediating Ligand Effects

Cancers ◽

10.3390/cancers13081816 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1816

Author(s):

Jessica Amarù ◽

Federica Barbieri ◽

Marica Arvigo ◽

Agnese Solari ◽

Adriana Bajetto ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Cells ◽

Intracellular Signaling ◽

Somatostatin Receptor ◽

Combined Treatment ◽

Primary Cultures ◽

Receptor Subtype ◽

Camp Accumulation ◽

Gh Secretion ◽

Somatostatin Receptor Ligands

First-generation somatostatin receptor ligands (fg-SRLs), such as octreotide (OCT), represent the first-line medical therapy in acromegaly. Fg-SRLs show a preferential binding affinity for somatostatin receptor subtype-2 (SST2), while the second-generation ligand, pasireotide (PAS), has high affinity for multiple SSTs (SST5 > SST2 > SST3 > SST1). Whether PAS acts via SST2 in somatotroph tumors, or through other SSTs (e.g., SST5), is a matter of debate. In this light, the combined treatment OCT+PAS could result in additive/synergistic effects. We evaluated the efficacy of OCT and PAS (alone and in combination) on growth hormone (GH) secretion in primary cultures from human somatotroph tumors, as well as on cell proliferation, intracellular signaling and receptor trafficking in the rat GH4C1 cell line. The results confirmed the superimposable efficacy of OCT and PAS in reducing GH secretion (primary cultures), cell proliferation, cAMP accumulation and intracellular [Ca2+] increase (GH4C1 cells), without any additive effect observed for OCT+PAS. In GH4C1 cells, co-incubation with a SST2-selective antagonist reversed the inhibitory effect of OCT and PAS on cell proliferation and cAMP accumulation, while both compounds resulted in a robust internalization of SST2 (but not SST5). In conclusion, OCT and PAS seem to act mainly through SST2 in somatotroph tumor cells in vitro, without inducing any additive/synergistic effect when tested in combination.

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