Comparison of Ciliary Targeting of Two Rhodopsin-Like GPCRs: Role of C-Terminal Localization Sequences in Relation to Cilium Type.

Primary cilia exhibit a distinct complement of proteins, including G-protein-coupled receptors (GPCRs) that mediate sensory and developmental signals. The localization of GPCRs to the ciliary membrane involves ciliary localization sequences (CLSs), but it is not known how CLSs might relate to cilium type. Here, we studied the localization of two rhodopsin (RHO)-like GPCRs, somatostatin receptor (SSTR3) and RHO, in three types of cilia, from inner medullary collecting duct (IMCD3) cells, hTERT-RPE1 cells (possessing pocket cilia), and rod photoreceptors (whose cilia grow into elaborate phototransductive outer segments). SSTR3 was localized specifically to all three types of cilia, whereas RHO showed more selectivity for the photoreceptor cilium. Focusing on C-terminal CLSs, we characterized a novel CLS in the SSTR3 C terminus, which was required for the robust ciliary localization of SSTR3. Replacing the C terminus of RHO with this SSTR3 CLS-enhanced ciliary localization, compared with full-length RHO in IMCD3 and hTERT-RPE1 cells. Addition of the SSTR3 CLS to the single transmembrane protein CD8A enabled ciliary localization. In hTERT-RPE1 cells, a partial SSTR3 CLS added to CD8A effected specific localization to the periciliary (pocket) membrane, demonstrating C-terminal localization sequence targeting to this domain. Using retinas from mice, including both sexes, we show that deletion of the C terminus of RHO reduced the rod outer segment localization and that addition of the SSTR3 C-terminal CLS to the truncated RHO partly rescued this mislocalization. Overall, the study details elements of the different C termini of SSTR3 and RHO that are major effectors in determining specificity of cilium (or pericilium) localization among different types of cilia.SIGNIFICANCE STATEMENT The localization of G-protein-coupled receptors to primary cilia is key to many types of signal transduction. After characterizing a novel C-terminal CLS in SSTR3, we investigated how SSTR3 and RHO localization to the cilium relates to C-terminal CLSs and to cilium type. We found that the SSTR3 C-terminal CLS was effective in three different types of cilia, but the RHO C terminus showed a clear localization preference for the highly elaborate photoreceptor cilium. When added to CD8A, part of the SSTR3 CLS promoted specific periciliary membrane localization in hTERT-RPE1 cells, demonstrating an effective CLS for this domain. Thus, we demonstrate that elements of the C termini of SSTR3 and RHO determine different localization patterns among different types of cilia.

HTR6 and SSTR3 targeting to primary cilia.

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.

Upregulated Expression of SSTR3 is Involved in Neuronal Apoptosis After Intracerebral Hemorrhage in Adult Rats.

Somatostatin which is a multifunctional growth hormone inhibitory neuropeptide shows diverse physiological effects, such as neurotransmission, cell growth, apoptosis, and endocrine signaling as well as exerts inhibitory effects on hormonal products and other secretory proteins. SSTR3 is a member of superfamily of somatostatin receptors (SSTR), which are G-protein-coupled plasma membrane receptors. Previous studies proved that SSTR3 regulates antiproliferative signaling and apoptosis in several cells. Here, we explored a potential role of SSTR3 in the regulation of neuronal apoptosis in the course of intracerebral hemorrhage (ICH). An ICH rat model was established and assessed by behavioral tests. We found SSTR3 was significantly upregulated surrounding the hematoma after ICH by Western blot and immunohistochemistry. Double immunofluorescence-manifested SSTR3 was strikingly increased in neurons, not astrocytes or microglia. Moreover, increasing SSTR3 level was found to be accompanied by the upregulation of p53, Bax, and active caspase-3 in vivo and in vitro studies. Furthermore, we detected that neuronal apoptosis marker active caspase-3 was co-localized with SSTR3 suggesting its potential role in neuronal apoptosis. In addition, in vitro study, revealed that SSTR3 knockdown specifically resulted in reducing neuronal apoptosis in PC12 cells, which further indicated that SSTR3 might exert its pro-apoptotic function on neuronal apoptosis. All our findings suggested that upregulated SSTR3 may be involved in neuronal apoptosis after ICH.

Discovery of substituted (4-phenyl-1H-imidazol-2-yl)methanamine as potent somatostatin receptor 3 agonists.

We report SAR studies on a novel non-peptidic somatostatin receptor 3 (SSTR3) agonist lead series derived from (4-phenyl-1H-imidazol-2-yl)methanamine. This effort led to the discovery of a highly potent low molecular weight SSTR3 agonist 5c (EC50=5.2 nM, MW=359). The results from molecular overlays of 5c onto the L-129 structure indicate good alignment, and two main differences of the proposed overlays of the antagonist MK-4256 onto the conformation of 5c lead to inversion of antagonism to agonism.

CPEB2 inhibits preeclampsia progression by regulating SSTR3 translation through polyadenylation.

AIMS: Trophoblast cell dysfunction is one of the important factors leading to preeclampsia (PE). Cytoplasmic polyadenylation element-binding 2 (CPEB2) has been found to be differentially expressed in PE patients, but whether it mediates PE process by regulating trophoblast cell function is unclear. METHODS: The expression of CPEB2 and somatostatin receptor 3 (SSTR3) was detected by quantitative real-time PCR, Western blot (WB) and immunofluorescence staining. Cell functions were analyzed by CCK-8 assay, EdU assay, flow cytometry and transwell assay. Epithelial-mesenchymal transition (EMT)-related protein levels were detected by WB. The interaction of CPEB2 and SSTR3 was confirmed by RIP assay, dual-luciferase reporter assay and PCR poly(A) tail assay. Animal experiments were performed to explore the effect of CPEB2 on PE progression in vivo, and the placental tissues of rat were used for H&E staining, immunohistochemical staining and TUNEL staining. RESULTS: CPEB2 was lowly expressed in PE patients. CPEB2 upregulation accelerated trophoblast cell proliferation, migration, invasion and EMT, while its knockdown had an opposite effect. CPEB2 bound to the CPE site in the 3'-UTR of SSTR3 mRNA to suppress SSTR3 translation through reducing poly(A) tails. Besides, SSTR3 overexpression suppressed trophoblast cell proliferation, migration, invasion and EMT, while its silencing accelerated trophoblast cell functions. However, these effects could be reversed by CPEB2 upregulation and knockdown, respectively. In vivo experiments, CPEB2 overexpression relieved histopathologic changes, inhibited apoptosis, promoted proliferation and enhanced EMT in the placenta of PE rat by decreasing SSTR3 expression. CONCLUSION: CPEB2 inhibited PE progression, which promoted trophoblast cell functions by inhibiting SSTR3 translation through polyadenylation.

Somatostatin-SSTR3-GSK3 modulates human T-cell responses by inhibiting OXPHOS.

Introduction: Somatostatin (SST) is a peptide hormone primarily synthesized in the digestive and nervous systems. While its impact on the endocrine system is well-established, accumulating evidence suggests a crucial role for SST and its analogues in modulating immune responses. Despite this, the precise mechanism through which SST regulates T cells has remained largely unknown. Methods: To elucidate the impact of SST on human T cells, we conducted a series of experiments involving cell culture assays, molecular analyses, and metabolic profiling. Human T cells were treated with SST, and various parameters including proliferation, cytokine production, and metabolic activities were assessed. Additionally, we employed pharmacological inhibitors and genetic manipulations to dissect the signaling pathways mediating SST's effects on T cells. Results: We showed that SST diminishes T-cell proliferation by influencing IL-2 production and T-cell mitochondrial respiration, while having no discernible impact on TCR-induced glycolysis. Our findings also identified that the regulatory influence of SST on T-cell responses and metabolism is contingent on its receptor, SSTR3. Moreover, we demonstrated that SST governs T-cell responses and metabolism by acting through the T-cell metabolic checkpoint GSK3. Discussion: Our study provides novel insights into the immunoregulatory function of SST in human T cells, highlighting the complex interplay between hormonal signaling and immune regulation. Understanding the molecular mechanisms underlying SST's effects on T cells may offer therapeutic opportunities for manipulating immune responses in various pathological conditions.

Beta cell primary cilia mediate somatostatin responsiveness via SSTR3.

Somatostatin is a paracrine modulator of insulin secretion and beta cell function with pleotropic effects on glucose homeostasis. The mechanism of somatostatin-mediated communication between delta and beta cells is not well-understood, which we address in this study via the ciliary somatostatin receptor 3 (SSTR3). Primary cilia are membrane organelles that act as signaling hubs in islets by virtue of their subcellular location and enrichment in signaling proteins such as G-protein coupled receptors (GPCRs). We show that SSTR3, a ciliary GPCR, mediates somatostatin suppression of insulin secretion in mouse islets. Quantitative analysis of calcium flux using a mouse model of genetically encoded beta cell-specific GCaMP6f calcium reporter shows that somatostatin signaling alters beta cell calcium flux after physiologic glucose stimulation, an effect that depends on endogenous SSTR3 expression and the presence of intact primary cilia on beta cells. Comparative in vitro studies using SSTR isoform antagonists demonstrate a role for SSTR3 in mediating somatostatin regulation of insulin secretion in mouse islets. Our findings support a model in which ciliary SSTR3 mediates a distinct pathway of delta-to-beta cell regulatory crosstalk and may serve as a target for paracrine modulation.

Identification of a Novel SSTR3 Full Agonist for the Treatment of Nonfunctioning Pituitary Adenomas.

Somatostatin receptor (SSTR) agonists have been extensively used for treating neuroendocrine tumors. Synthetic therapeutic agonists showing selectivity for SSTR2 (Octreotide) or for SSTR2 and SSTR5 (Pasireotide) have been approved for the treatment of patients with acromegaly and Cushing's syndrome, as their pituitary tumors highly express SSTR2 or SSTR2/SSTR5, respectively. Nonfunctioning pituitary adenomas (NFPAs), which express high levels of SSTR3 and show only modest response to currently available SSTR agonists, are often invasive and cannot be completely resected, and therefore easily recur. The aim of the present study was the evaluation of ITF2984, a somatostatin analog and full SSTR3 agonist, as a new potential treatment for NFPAs. ITF2984 shows a 10-fold improved affinity for SSTR3 compared to Octreotide or Pasireotide. Molecular modeling and NMR studies indicated that the higher affinity for SSTR3 correlates with a higher stability of a distorted ß-I turn in the cyclic peptide backbone. ITF2984 induces receptor internalization and phosphorylation, and triggers G-protein signaling at pharmacologically relevant concentrations. Furthermore, ITF2984 displays antitumor activity that is dependent on SSTR3 expression levels in the MENX (homozygous mutant) NFPA rat model, which closely recapitulates human disease. Therefore, ITF2984 may represent a novel therapeutic option for patients affected by NFPA.

Ciliary neuropeptidergic signaling dynamically regulates excitatory synapses in postnatal neocortical pyramidal neurons.

Primary cilia are compartmentalized sensory organelles present on the majority of neurons in the mammalian brain throughout adulthood. Recent evidence suggests that cilia regulate multiple aspects of neuronal development, including the maintenance of neuronal connectivity. However, whether ciliary signals can dynamically modulate postnatal circuit excitability is unknown. Here we show that acute cell-autonomous knockdown of ciliary signaling rapidly strengthens glutamatergic inputs onto cultured rat neocortical pyramidal neurons and increases spontaneous firing. This increased excitability occurs without changes to passive neuronal properties or intrinsic excitability. Further, the neuropeptide receptor somatostatin receptor 3 (SSTR3) is localized nearly exclusively to excitatory neuron cilia both in vivo and in culture, and pharmacological manipulation of SSTR3 signaling bidirectionally modulates excitatory synaptic inputs onto these neurons. Our results indicate that ciliary neuropeptidergic signaling dynamically modulates excitatory synapses and suggest that defects in this regulation may underlie a subset of behavioral and cognitive disorders associated with ciliopathies.

Somatostatin Serves a Modulatory Role in the Mouse Olfactory Bulb: Neuroanatomical and Behavioral Evidence.

Somatostatin (SOM) and somatostatin receptors (SSTR1-4) are present in all olfactory structures, including the olfactory bulb (OB), where SOM modulates physiological gamma rhythms and olfactory discrimination responses. In this work, histological, viral tracing and transgenic approaches were used to characterize SOM cellular targets in the murine OB. We demonstrate that SOM targets all levels of mitral dendritic processes in the OB with somatostatin receptor 2 (SSTR2) detected in the dendrites of previously uncharacterized mitral-like cells. We show that inhibitory interneurons of the glomerular layer (GL) express SSTR4 while SSTR3 is confined to the granule cell layer (GCL). Furthermore, SOM cells in the OB receive synaptic inputs from olfactory cortical afferents. Behavioral studies demonstrate that genetic deletion of SSTR4, SSTR2 or SOM differentially affects olfactory performance. SOM or SSTR4 deletion have no major effect on olfactory behavioral performances while SSTR2 deletion impacts olfactory detection and discrimination behaviors. Altogether, these results describe novel anatomical and behavioral contributions of SOM, SSTR2 and SSTR4 receptors in olfactory processing.

Discovery of MK-1421, a Potent, Selective sstr3 Antagonist, as a Development Candidate for Type 2 Diabetes.

The imidazolyl-tetrahydro-ß-carboline class of sstr3 antagonists have demonstrated efficacy in a murine model of glucose excursion and may have potential as a treatment for type 2 diabetes. The first candidate in this class caused unacceptable QTc interval prolongation in oral, telemetrized cardiovascular (CV) dogs. Herein, we describe our efforts to identify an acceptable candidate without CV effects. These efforts resulted in the identification of (1R,3R)-3-(4-(5-fluoropyridin-2-yl)-1H-imidazol-2-yl)-1-(1-ethyl-pyrazol-4-yl)-1-(3-methyl-1,3,4-oxadiazol-3H-2-one-5-yl)-2,3,4,9-tetrahydro-1H-ß-carboline (17e, MK-1421).

Investigation of Cardiovascular Effects of Tetrahydro-ß-carboline sstr3 antagonists.

Antagonism of somatostatin subtype receptor 3 (sstr3) has emerged as a potential treatment of Type 2 diabetes. Unfortunately, the development of our first preclinical candidate, MK-4256, was discontinued due to a dose-dependent QTc (QT interval corrected for heart rate) prolongation observed in a conscious cardiovascular (CV) dog model. As the fate of the entire program rested on resolving this issue, it was imperative to determine whether the observed QTc prolongation was associated with hERG channel (the protein encoded by the human Ether-à-go-go-Related Gene) binding or was mechanism-based as a result of antagonizing sstr3. We investigated a structural series containing carboxylic acids to reduce the putative hERG off-target activity. A key tool compound, 3A, was identified from this SAR effort. As a potent sstr3 antagonist, 3A was shown to reduce glucose excursion in a mouse oGTT assay. Consistent with its minimal hERG activity from in vitro assays, 3A elicited little to no effect in an anesthetized, vagus-intact CV dog model at high plasma drug levels. These results afforded the critical conclusion that sstr3 antagonism is not responsible for the QTc effects and therefore cleared a path for the program to progress.

The Discovery of MK-4256, a Potent SSTR3 Antagonist as a Potential Treatment of Type 2 Diabetes.

A structure-activity relationship study of the imidazolyl-ß-tetrahydrocarboline series identified MK-4256 as a potent, selective SSTR3 antagonist, which demonstrated superior efficacy in a mouse oGTT model. MK-4256 reduced glucose excursion in a dose-dependent fashion with maximal efficacy achieved at doses as low as 0.03 mg/kg po. As compared with glipizide, MK-4256 showed a minimal hypoglycemia risk in mice.