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. 2013 Sep 6;288(36):25689-25700.
doi: 10.1074/jbc.M113.496414. Epub 2013 Aug 2.

Agonist-biased trafficking of somatostatin receptor 2A in enteric neurons

Peishen Zhao  1 , Meritxell Canals  1 , Jane E Murphy  2 , Diana Klingler  3 , Emily M Eriksson  4 , Juan-Carlos Pelayo  2 , Markus Hardt  3 , Nigel W Bunnett  5 , Daniel P Poole  6
Affiliations

Agonist-biased trafficking of somatostatin receptor 2A in enteric neurons

Peishen Zhao et al. J Biol Chem. .

Abstract

Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with β-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and β-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/β-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from β-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained β-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.

Keywords: Arrestin; Endocytosis; Endothelin-converting Enzyme; Neurons; Neuropeptide; Receptor Recycling; Somatostatin.

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Figures

FIGURE 1.
FIGURE 1.
SSTR2A endocytosis is concentration-dependent in myenteric neurons. A, SSTR2A-IR was localized to the plasma membrane of the soma and neurites (arrowheads) in untreated nitric oxide synthase-IR neurons (0 min). SSTR2A-postive pixels above threshold are shown in the right column. SST-14 (1–1000 nm, 60 min, 4 °C, wash, 30 min recovery, 37 °C) induced a concentration-dependent internalization of SSTR2A-IR (arrows). Scale bars = 10 μm. B, quantitative image analysis of intracellular SSTR2A-IR (INT) in response to graded concentrations of SST-14. C, comparison of relative intracellular SSTR2A following internalization to different SSTR2A agonists (all 100 nm). Receptor distribution at 30 and 120 min recovery times are shown. *, p < 0.1; ***, p < 0.001 to 0 min control.
FIGURE 2.
FIGURE 2.
Agonists induce endocytosis of SSTR2A in myenteric neurons. Photomicrographs show the localization of SSTR2A-IR in nitric oxide synthase-IR myenteric neurons. The threshold image shows SSTR2A-positive staining in white. Arrowheads show SSTR2A-IR at the plasma membrane, and arrows show internalized SSTR2A-IR. Column graphs show the proportion of SSTR2A-IR in the soma at the plasma membrane (PM) and internalized (INT). Whole-mounts were incubated with agonists, washed, and recovered for the indicated times at 37 °C. A, SST-14-induced trafficking of SSTR2A-IR. In untreated neurons (0 min), SSTR2A-IR was localized to the plasma membrane of the soma and neurites. After incubation with SST-14, washing, and recovery, SSTR2A-IR internalized (30 min), and then recycled (60–120 min). NOS, nitric oxide synthase. B, SST-28 also induced SSTR2A-IR endocytosis, but the receptor did not fully recycle after 120 min. C, L-054,264 stimulated transient SSTR2A-IR internalization (30 min) followed by a complete recycling within 60 min. D and E, octreotide and vapreotide induced sustained internalization with no detectable recycling. Scale bars = 10 μm. ***, p < 0.001 versus 0 min.
FIGURE 3.
FIGURE 3.
Agonists induce dynamin- and clathrin-dependent endocytosis of SSTR2A in myenteric neurons. A, octreotide stimulated marked SSTR2A-IR endocytosis after 30 min (arrow, top row), which was prevented by dynasore and sucrose (arrowheads). Scale bars = 10 μm. NOS, nitric oxide synthase. B, column graph indicating that dynasore and sucrose significantly inhibit octreotide-stimulated endocytosis of SSSTR2A-IR. PM, plasma membrane; INT, internalized. ***, p < 0.001 to vehicle control.
FIGURE 4.
FIGURE 4.
ECE-1 regulates SSTR2A recycling in myenteric neurons. A, in neurons treated with the ECE-1 inhibitor SM-19712 (10 μm), SST-14 caused internalization of SSTR2A-IR, which was retained even after 120-min recovery (arrows). In vehicle-treated neurons, SSTR2A-IR was fully recycled after 120-min recovery (arrowheads). NOS, nitric oxide synthase. B, column graph showing quantitative analysis of receptor distribution. PM, plasma membrane-associated; INT, internalized; SM, SM-19712. ***, p < 0.001 to vehicle control. C, bafilomycin A1 similarly inhibited SSTR2A-IR recycling at 120 min (arrow). D, ECE-1-IR was present in SSTR2A-IR myenteric neurons of the rat distal colon. In untreated preparations (0 min), there was no colocalization of ECE-1-IR and SSTR2A-IR (arrowheads). SST-28 induced trafficking of SSTR2A-IR to ECE-1-IR intracellular structures within the soma (arrows). Scale bars = 10 μm.
FIGURE 5.
FIGURE 5.
SSTR2A traffics to the TGN and recycles in myenteric neurons. A, octreotide induced SSTR2A-IR trafficking from the plasma membrane (arrowheads, 0 min) to TGN38-IR structures (arrows, 30 min). B, brefeldin A disrupted the TGN and caused a redistribution of internalized SSTR2A-IR to multiple vesicles. C, brefeldin A did not affect SST-14-stimulated endocytosis of SSTR2A-IR (arrow, 30 min) but caused intracellular retention of SSTR2A-IR up to 120 min (arrows). Scale bars = 10 μm. NOS, nitric oxide synthase. D, column graph showing quantitative analysis of receptor distribution. PM, plasma membrane-associated; INT, internalized; BFA, brefeldin A. ***, p < 0.001 to vehicle control.
FIGURE 6.
FIGURE 6.
SSTR2A trafficking in At-T20 cells. A, under basal conditions (0 min), SSTR2A-IR was at the plasma membrane (red, arrowhead), and ECE-1d-GFP was intracellular. SST-28 stimulated trafficking of SSTR2A-IR to intracellular locations that colocalized with ECE-1d-GFP (30–120 min, arrows). B, ECE-1d-GFP partially colocalized with Golgin97 in the Golgi apparatus (arrows). C, SST-14, SST-28 and L-054,264 and vapreotide stimulated internalization of SSTR2A-IR (30 min). SSTR2A-IR was recycled 60 min after L-054,264, 120 min after SST-14 and SST-28, but not at all after vapreotide. Scale bars = 5 μm.
FIGURE 7.
FIGURE 7.
SSTR2A interacts with the ECE-1a and ECE-1d isoforms. SSTR2A-RLuc8 and ECE-1a-YFP or ECE-1d-YFP were coexpressed in HEK cells. A, stimulation with SST-14, SST-28 and octreotide (100 nm) resulted in a significant reduction in BRET signal between SSTR2A-RLuc8 and the plasma membrane-associated ECE-1a-YFP isoform, indicating a loss of interaction between these two proteins. B, in contrast, agonist stimulation gave rise to a significant increase in BRET signal between SSTR2A-RLuc8 and the endosome-associated ECE-1d-YFP isoform, indicating increased interaction. **, p < 0.01; ***, p < 0.001.
FIGURE 8.
FIGURE 8.
ECE-1 degrades SST-14 at endosomal pH. SST-14 (A and B) and SST-28 (E and F) were incubated with ECE-1 at endosomal (pH 5.5) and extracellular (pH 7.4) pH. Degradation and formation of products was assessed by proteinase activity labeling employing 18O-enriched water. A–C, SST-14 (1637 Da, black) was degraded at Thr10-Phe11, and the products remained disulfide-linked, which resulted in a mass gain of 18 Da because of water incorporation (1655, blue). ECE-1 then cleaved at Phe6-Phe7, forming a second linked product (1093, orange). Degradation was far more rapid at pH 5.5 than pH 7.4. E–G, at pH 5.5, SST-28 (3147 Da, black) was degraded at Thr24-Phe25, and the products remained disulfide-linked, which resulted in a gain of mass of 18 Da because of water incorporation (3165, blue). ECE-1 then cleaved at Phe20-Phe21, forming a second linked product (2456, orange). Degradation of SST-28 was negligible at pH 7.4. D, comparison of the rate of degradation of SST-14 and SST-28 at pH 5.5.
FIGURE 9.
FIGURE 9.
ECE-1 regulates SSTR2A/β-arrestin interactions. A, SSTR2A-RLuc8 and β-arrestin-YFP were coexpressed in HEK cells. Agonist stimulation induced BRET, which is indicative of SSTR2A-RLuc8 and β-arrestin-YFP interactions. B and C, effects of graded concentrations of SST-14, SST-28, and octreotide on the interaction between SSTR2A-RLuc8 and β-arrestin1-YFP (B) and β-arrestin2-YFP (C). D, time course of SSTR2A-RLuc8 and β-arrestin2-YFP interaction in the continued presence of SST-14 (left panel), SST-28 (center panel), or octreotide (right panel). In vehicle-treated cells, SST-14- and SST-28-induced BRET declined, whereas octreotide-induced BRET was sustained. SM-19712 maintained SST-14- and SST-28-induced BRET. E, SSTR2A-RLuc8 and β-arrestin2-YFP interaction measured at various times after stimulation with SST-14 (left panel), SST-28 (center panel), or octreotide (right panel). In vehicle-treated cells, SST-14- and SST-28-induced BRET declined after washing, whereas octreotide-induced BRET was sustained. SM-19712 maintained SST-14- and SST-28-induced BRET. *, p < 0.001 to vehicle control.
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