Pathophysiology of GPCR Homo- and Heterodimerization: Special Emphasis on Somatostatin Receptors

G-protein coupled receptors (GPCRs) are cell surface proteins responsible for translating >80% of extracellular reception to intracellular signals. The extracellular information in the form of neurotransmitters, peptides, ions, odorants etc is converted to intracellular signals via a wide variety of effector molecules activating distinct downstream signaling pathways. All GPCRs share common structural features including an extracellular N-terminal, seven-transmembrane domains (TMs) linked by extracellular/intracellular loops and the C-terminal tail. Recent studies have shown that most GPCRs function as dimers (homo- and/or heterodimers) or even higher order of oligomers. Protein-protein interaction among GPCRs and other receptor proteins play a critical role in the modulation of receptor pharmacology and functions. Although ~50% of the current drugs available in the market target GPCRs, still many GPCRs remain unexplored as potential therapeutic targets, opening immense possibility to discover the role of GPCRs in pathophysiological conditions. This review explores the existing information and future possibilities of GPCRs as tools in clinical pharmacology and is specifically focused for the role of somatostatin receptors (SSTRs) in pathophysiology of diseases and as the potential candidate for drug discovery.Pharmaceutical Sciences, Faculty ofReviewedFacult

δ-Opioid Receptor and Somatostatin Receptor-4 Heterodimerization: Possible Implications in Modulation of Pain Associated Signaling

<div><p>Pain relief is the principal action of opioids. Somatostatin (SST), a growth hormone inhibitory peptide is also known to alleviate pain even in cases when opioids fail. Recent studies have shown that mice are prone to sustained pain and devoid of analgesic effect in the absence of somatostatin receptor 4 (SSTR4). In the present study, using brain slices, cultured neurons and HEK-293 cells, we showed that SSTR4 and δ-Opioid receptor (δOR) exist in a heteromeric complex and function in synergistic manner. SSTR4 and δOR co-expressed in cortical/striatal brain regions and spinal cord. Using cultured neuronal cells, we describe the heterogeneous complex formation of SSTR4 and δOR at neuronal cell body and processes. Cotransfected cells display inhibition of cAMP/PKA and co-activation of SSTR4 and δOR oppose receptor trafficking induced by individual receptor activation. Furthermore, downstream signaling pathways either associated with withdrawal or pain relief are modulated synergistically with a predominant role of SSTR4. Inhibition of cAMP/PKA and activation of ERK1/2 are the possible cellular adaptations to prevent withdrawal induced by chronic morphine use. Our results reveal direct intra-membrane interaction between SSTR4 and δOR and provide insights for the molecular mechanism for the anti-nociceptive property of SST in combination with opioids as a potential therapeutic approach to avoid undesirable withdrawal symptoms.</p></div

Regulation of cAMP/PKA signaling pathways in receptor and agonist dependent manner.

<p>(A) Receptor coupling to adenylyl cyclase. SST, L-803087 and SB-205607 displayed significant inhibition of FSK stimulated cAMP in comparison to control. Data is representative of three independent experiments and presented as % inhibition upon treatment as indicated. (B) Concentration dependent inhibition of cAMP in HEK-293 cells. Treatment of cells with FSK alone was taken as 0% inhibition and treatment with forskolin and SST (1 µM) was considered as 100% inhibition. Note the significant increase in the efficiency of cAMP inhibition upon treatment with SST (10⁻¹²–10⁻⁶ M) in combination with SB-205607 (10 nM). (C and D) PKA phosphorylation in mono-and/or cotransfected cells. HEK-293 cells expressing SSTR4 and/or δOR were treated for 15 min at 37°C as indicated and cell lysate prepared was subjected to western blot analysis. In monotransfected cells, the status of phospho-PKA was comparable to basal upon receptor specific activation (C). In cotransfected cells, significant inhibition of PKA phosphorylation was observed which was further enhanced upon combined agonist treatment as indicated (D). Densitometric analysis for phospho-PKA was performed by using β-actin or total as loading control and data analysis was done by using ANOVA and <i>post hoc</i> Dunnett’s to compare against basal level (*, <i>p</i><0.05).</p

Changes in AKT phosphorylation are independent to PI3K activation.

<p>(<b>A</b>) HEK-293 cells expressing SSTR4 and /or δOR were treated as indicated for 15 min at 37°C and cell lysate prepared post treatment was subjected to western blot analysis to detect phospho and total-PI3K and AKT expression. The status of phospho-PI3K remained comparable to basal upon treatment with SST or SSTR specific agonist in cells expressing SSTR4. In contrast, δOR monotransfectant were devoid of phospho-PI3K expression with or without receptor specific treatment. (<b>B</b>) Significant activation of phospho-PI3K was observed in cotransfected cells at the basal level which remained comparable upon agonist treatments as indicated. (<b>C</b>) δOR monotransfected cells displayed significant increase in phospho-AKT expression upon treatment with receptor specific agonist in comparison to control, whereas, no discernible changes in phospho-AKT were observed in SSTR4 monotransfected cells. (<b>D</b>) In cotransfected cells, SSTR4 activation with SST or L-803087 alone or in combination with SB-205607 significantly enhanced AKT phosphorylation, with pronounced effect upon combined agonist treatment. Histograms illustrate densitometry for the blots for respective panels using β-actin or total as loading control. Data analysis was done by using ANOVA and <i>post hoc</i> Dunnett’s to compare against basal level (*, <i>p</i><0.05).</p

Colocalization and Co-Immunoprecipitation of SSTR4 and δOR in rat brain and spinal cord.

<p>(<b>A</b>) Representative confocal photomicrographs illustrating the colocalization of SSTR4 and δOR in rat brain cerebral cortex, striatum and spinal cord. 30 µm sections from brain and spinal cord were incubated with SSTR4 and δOR specific primary antibodies, followed by incubation with secondary antibodies. The expression of δOR (red), SSTR4 (green) and colocalization is identified in orange yellow color. Note, all δOR positive neurons colocalized with SSTR4 in the three brain regions including cortex, striatum and ventral and dorsal horn of spinal cord respectively. In addition, SSTR4 positive neurons devoid of colocalization are indicated by green arrows in respective panel. In spinal cord, nerve fibres positive to δOR were devoid of colocalization with SSTR4 (red arrows spinal cord bottom panel). Astrics (*) indicate astrocytes in cortical and striatal brain sections. (Scale bar  =  10 µm for upper panels and 100 µm for bottom panel; n  =  3). (<b>B</b>) Expression of δOR in SSTR4 immunoprecipitate prepared from rat brain and spinal cord. Membrane preparation from cortex, striatum and spinal cord tissue lysate was treated with SSTR4 specific agonist (L-803087), δOR specific agonist (SB-205607) alone or in combination for 30 min at 37°C. Following treatments, tissue lysate was immunoprecipitated with SSTR4 antibody and immunoblotted for δOR specific antibody as described in Methods section. The expression of δOR at approximately ∼90 kDa indicates that δOR and SSTR4 exist in a complex in brain.</p

Co-activation of SSTR4 and δOR retained receptors expression at the cell surface.

<p>(<b>A</b>) Representative confocal photomicrographs illustrating membrane and intracellular expression of SSTR4 and δOR in non-permeabilized (NP) and permeabilized (P) HEK-293 cells. Indicated color in red, green and yellow/orange (merged images) represents the expression of δOR, SSTR4 and colocalization respectively. In cotransfectants, the activation of SSTR4 (upper panel right) or δOR (lower panel left) preferentially promotes receptor internalization which was blocked upon combined agonist treatment. Histogram in panels <b>B and C</b> showed the quantification of receptor expression in non-permeabilized (NP) and permeabilized (P) conditions respectively performed by using NIH Image J software. Note the significant loss in expression of δOR (B, red histogram) and SSTR4 (B, green histogram) at cell surface upon treatment with receptor specific agonists as indicated. Intracellular expression of δOR (C, red histogram) and SSTR4 (C, green histogram) was significantly enhanced following treatments with SB-205607 and SST alone without having any discernible effects in combination (*, <i>p</i><0.05). Scale bar  =  10 µm.</p

Receptors mediated changes in ERK1/2 and ERK5 phosphorylation.

<p>(<b>A</b>) HEK-293 cells expressing SSTR4 and /or δOR were treated as indicated for 15 min at 37°C and cell lysate was subjected to Western blot analysis. In SSTR4 monotransfected cells, SST and SSTR4 agonist resulted in inhibition of phospho-ERK1/2 when compared to control, whereas, δOR activation was without any significant effect on ERK1/2 phosphorylation. (<b>B</b>) In cotransfected cells, SSTR4 activation displayed increased ERK1/2 phosphorylation. In presence of δOR agonist alone or in combination with SST or L-803087, ERK1/2 activation was decreased although remained significantly higher than control. (<b>C</b>) In monotransfected cells, SSTR4 activation resulted in enhanced phospho-ERK5 expression, whereas, no change in the phospho-ERK5 levels was observed upon δOR activation. (<b>D</b>) In cotransfected cells, increased ERK5 phosphorylation was observed upon SSTR4 or δOR activation alone or in combination. Note that the combined agonist treatment had pronounced effect on ERK5 phosphorylation. Densitometric analysis for the blots (in Panels A-D) was performed by using β-actin or total as loading control and data analysis was done by using ANOVA and <i>post hoc</i> Dunnett’s to compare against basal level (*, <i>p</i><0.05).</p

Somatostatin preserved blood brain barrier against cytokine induced alterations: possible role in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory neurological disorder associated with demyelination, impaired blood brain barrier (BBB), axonal damage and neuronal loss. In the present study, we measured somatostatin (SST) and tumor necrosis factor-α (TNF-α) like immunoreactivity in CSF samples from MS and non-MS patients. We also examined the role of SST in cytokines and lipopolysaccharide (LPS)-induced damage to the BBB using human brain endothelial cells in culture. Most of the cerebrospinal fluid (CSF) samples studied from definite MS patients exhibited lower somatostatin (SST)-like immunoreactivity and higher expression of TNF-α in comparison to non-MS patients. Treatment of cells with cytokines and LPS blocked SST secretion and decreased SST expression. Human brain endothelial cells expressed all five somatostatin receptors (SSTRs) with increased expression of SSTR2 and 4 upon treatment with cytokines and LPS. Cytokines and LPS-induced disruption of the tight junction proteins Zonula occludens (ZO-1) organization was restored in presence of SST, SSTR2 or SSTR4 selective agonists. Furthermore, inflammation induced changes in extracellular signal-regulated kinases (ERK1/2 and ERK5) signaling and altered expression of endothelial and inducible nitric oxide synthase are modulated in presence of SST. These data indicate that decreased levels of SST contribute to failure of the BBB in MS