Dissecting G Protein-coupled Receptor Signaling Pathways with Membrane-permeable Blocking Peptides

Abstract

To determine the intracellular signaling mechanism of the 5-HT 2C receptor endogenously expressed in choroid plexus epithelial cells, we implemented a strategy of targeted disruption of protein-protein interactions. This strategy entails the delivery of conjugated membrane-permeable peptides that disrupt domain interaction at specific steps in the signaling cascade. As proof of concept, two peptides targeted against receptor-G protein interaction domains were examined. Only G q CT, which targets the receptor-G q protein interacting domain, disrupted 5-HT 2C receptor-mediated phosphatidylinositide hydrolysis. G s CT, targeting the receptor-G s protein, disrupted ␤2 adrenergic receptor-mediated activation of cAMP but not 5-HT 2C receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLC␤1M, mimicking the domain of phospholipase C␤1 (PLC␤1) interacting with active G␣ q , also blocked 5-HT 2C receptor activation. In contrast, peptides PLC␤2M and Phos that bind to and sequester free G␤␥ subunits were ineffective at blocking 5-HT 2C receptormediated phosphoinositol turnover. However, both peptides disrupted G␤␥-mediated ␣ 2A adrenergic receptor activation of mitogen-activated protein kinase. These results provide the first direct demonstration that active G␣ q subunits mediate endogenous 5-HT 2C receptor activation of PLC␤ and that G␤␥ subunits released from G␣ q heterotrimeric proteins are not involved. Comparable results were obtained with metabotropic glutamate receptor 5 expressed in astrocytes. Thus, conjugated, membrane-permeable peptides are effective tools for the dissection of intracellular signals.The 5-HT 2 receptor family consists of three members, 5-HT 2A , 5-HT 2B , and 5-HT 2C . All three receptors belong to the G protein-coupled serpentine receptor superfamily. Their pharmacological profiles are very similar, leading to difficulty in defining their functional roles. 5-HT 2 receptors have been implicated in behaviors such as sleep, feeding, aggression, pain, and anxiety and are thought to play a role in a number of central nervous system disorders including affective disease, schizophrenia, and epilepsy (1). In addition, 5-HT 2 receptors may play a major role in mediating the actions of hallucinogenic drugs (2) as well as antipsychotic drugs (3, 4). Mice expressing nonfunctional 5-HT 2C receptors exhibit epileptic and obese phenotypes (5, 6), suggesting that these receptors play a crucial role in moderating central nervous system function.Expression of the 5-HT 2C receptor is exceptionally high in the choroid plexus (7,8), where it plays a role in the regulation of production and composition of cerebrospinal fluid (9 -11). Initial studies of 5-HT 2C receptor signaling showed that these receptors activate the downstream intracellular effector, phospholipase C␤ (PLC␤) 1 resulting in the hydrolysis of phosphatidylinositol-4,5-bisphosphate into inositol-1,4,5-triphosphate and diacylglycerol (12). In addition, activation of the 5-HT 2C receptor has been observed to release arachidonic acid (13...

Histamine H3-receptor signaling in cardiac sympathetic nerves: Identification of a novel MAPK-PLA2-COX-PGE2-EP3R pathway

“…The Gβγ i subunit is known to inhibit Ca 2+ entry via N-type Ca 2+ -channels [22;29]. Thus, we hypothesized that signaling via the Gβγ i subunit could represent a common mechanism of action in the attenuation of NE exocytosis by H 3 R and EP 3 R. Indeed, we found that the membrane-permeable Gβγ-blocking peptide [23], which prevented the imetit-induced MAPK activation in SH-SY5Y-H 3 cells, significantly attenuated the inhibition of K + -induced NE exocytosis elicited by imetit and sulprostone in cardiac sympathetic nerve endings. This suggests that neuronal Ca 2+ -channel blockade via the Gβγ isubunit plays a role in the H 3 R-and EP 3 R-mediated inhibition of NE exocytosis and that MAPK is an important upstream step in this pathway.…”

“…The Gβγ i subunit is known to inhibit Ca 2+ entry [22]; thus, the Gβγ i subunit could play a role in the H 3 R-mediated attenuation of NE exocytosis. To test this hypothesis we used a phosducin-like membrane-permeable Gβγ-blocking peptide [23]. As shown in Figure 8, the H 3 R agonist imetit (100 nM) and the EP 3 R agonist sulprostone (300 nM) each caused a ~60% attenuation of K + -induced NE exocytosis from guinea-pig heart synaptosomes.…”

Role of Gq protein in behavioral effects of the hallucinogenic drug 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane

“…Inositol triphosphate promotes intracellular calcium mobilization, while diacylglycerol stimulates the activity of protein kinase C (Exton, 1996). 5-Hydroxytryptamine 2A (5-HT) 2A receptors are a subtype of the 5-HT2 subfamily of receptors, known to stimulate the PLC pathway via Gα q (Chang et al, 2000). 5HT 2A receptors are widely distributed in brain, including cortex, caudate nucleus, olfactory tubercle, nucleus accumbens, and hippocampus.…”

Involvement of Both Gq/11 and Gs Proteins in Gonadotropin-releasing Hormone Receptor-mediated Signaling in LβT2 Cells

“…Disrupting this protein-protein interaction, by introducing one of the binding domains into cell, can thus block specific pathways. Previous studies (34,37,38) have shown that the GPCR/G protein interaction can be disrupted in vitro by peptides derived from the C terminus of the G protein. This approach to analyze signaling has been limited, however, as most peptides do not readily penetrate the cell membrane.…”

“…We also generated peptides to inhibit G␣ s and G␤␥ signaling as controls. These inhibitory peptides are based on published sequences (34). The peptides were expressed as TAT fusion proteins to allow internalization.…”