Solubilization of G protein-coupled receptors: a convenient strategy to explore lipid-receptor interaction.

G protein-coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across cell membranes and are major drug targets. Since GPCRs are integral membrane proteins, their structure and function are modulated by membrane lipids. In particular, membrane cholesterol is an important lipid in the context of GPCR function. Solubilization of integral membrane proteins is a process in which the proteins and lipids in native membranes are dissociated in the presence of a suitable amphiphilic detergent. Interestingly, solubilization offers a convenient approach to monitor lipid-receptor interaction as it results in differential extents of lipid solubilization, thereby allowing to assess the role of specific lipids on receptor function. In this review, we highlight how this solubilization strategy is utilized to decipher novel information about the structural stringency of cholesterol necessary for supporting the function of the serotonin(1A) receptor. We envision that insight in GPCR-lipid interaction would result in better understanding of GPCR function in health and disease.

Stereospecific requirement of cholesterol in the function of the serotonin1A receptor.

The serotonin1A receptor is an important member of the G protein-coupled receptor (GPCR) family. It is involved in the generation and modulation of a variety of cognitive and behavioral functions and serves as a drug target. Previous work from our laboratory has established the sensitivity of the function of the serotonin1A receptor to membrane cholesterol. Solubilization of the hippocampal serotonin1A receptor utilizing the zwitterionic detergent CHAPS is accompanied by loss of cholesterol and results in reduction in specific ligand binding. Replenishment of cholesterol to solubilized membranes restores specific ligand binding to the receptor. We utilized this strategy of sterol replenishment of solubilized membranes to explore the stereospecific stringency of cholesterol for receptor function. We used two stereoisomers of cholesterol, ent-cholesterol (enantiomer of cholesterol) and epi-cholesterol (a diastereomer of cholesterol), for this purpose. Importantly, we show here that while ent-cholesterol could replace cholesterol in supporting receptor function, epi-cholesterol could not. These results imply that the requirement of membrane cholesterol for the serotonin1A receptor function is diastereospecific, yet not enantiospecific. Our results extend and help define specificity of the interaction of membrane cholesterol with the serotonin1A receptor, and represent the first report utilizing ent-cholesterol to examine stereospecificity of GPCR-cholesterol interaction.

Mass spectrometric characterization of recombinant rat 5-hydroxytryptamine receptor 1A (5-HT(1A) R) expressed in tsA201 human embryonic kidney cells.

The 5-hydroxytryptamine 1A receptor (serotonin 1A receptor; 5-HT(1A) R) is involved in a large series of brain functions, and roles in anxiety, depression, and cognition have been reported. So far, published information on mass spectrometrical characterization of 5-HT(1A) R is limited to the presence of two 5-HT(1A) R peptides in rat's whole brain as observed by in-solution digestion followed by LC-MS/MS. Knowledge about the protein sequence and PTMs, however, would have implications for generation of specific antibodies and designing studies on the 5-HT(1A) R at the protein level. A rat recombinant 5-HT(1A) R was extracted from the tsA201 cell line, run using several gel-based principles with subsequent in-gel digestion with several proteases, chymotrypsin, trypsin, AspN, proteinase K, and pepsin followed by nano-LC-ESI-MS/MS analysis on a high capacity ion trap and an LTQ Orbitrap Velos. Using two search engines, Mascot and Modiro™, the recombinant 5-HT(1A) R was identified showing 94.55% sequence coverage. A single phosphorylation at S301 was identified and verified by phosphatase treatment and a series of amino acid substitutions were detected. Characterization of 5-HT(1A) R, a key player of brain functions and neurotransmission, was shown and may enable generation of specific antibodies, design of future, and interpretation of previous studies in the rat at the protein level.

Unión al autorreceptor 5-HT1A de la serotonina en el núcleo dorsal del rafe en muestras de tejido de víctimas de suicidio deprimidas / Serotonin-1A autoreceptor binding in the dorsal raphe nucleus of depressed suicides

a disfunción serotoninérgica está presente en los trastornos del humor y el suicidio. Los autorreceptores somatodrendríticos 5-HT1A del tronco cerebral regulan la descarga de neuronas serotoninérgicas, pero los estudios efectuados sobre la unión a los autorreceptores en el núcleo dorsal del rafe (NDR) en muestras de tejidos obtenidas en víctimas de suicidio deprimidas describen resultados conflictivos. Tratamos de determinar: 1) la distribución anatómica de la unión al receptor 5-HT1A en el NDR de muestras de tejidos obtenidas de víctimas deprimidas de suicidio y de individuos de control sanos, desde un punto de vista psiquiátrico, fallecidos por causas naturales, y 2) si las diferencias de sexo en la unión a 5-HT1A en el NDR contribuyen a las diferencias observadas en las muestras entre víctimas deprimidas de suicidio y los de control. Se analizaron las autorradiografías cuantitativas del receptor de [3H]8-hidroxi-2-(di-n-propil) aminotetralina ([3H]8-OH-DPAT) en las secciones de tejido post mórtem, obtenidas previamente, que contenían el NDR de víctimas de suicidio libres de fármacos/drogas (n=10) y de controles emparejados (n=10). En el NDR íntegro de los suicidas deprimidos, comparado con los controles, se observó una menor unión total al receptor (fmol/mg de tejido×mm3) (p<0,05). Se observaron diferencias de grupo a lo largo de la extensión rostrocaudal del NDR para la unión transversal a 5-HT1A (fmol/mg de tejido) y la unión al receptor (fmol/mg×mm3, p<0,05). Comparado con controles, en el NDR de suicidas deprimidos la unión transversal a 5-HT1A fue mayor rostralmente y menor, caudalmente. Las diferencias entre suicidas deprimidos y controles estuvieron presentes en hombres y mujeres, aunque en estas se detectó una mayor unión que en hombres. La menor unión al autorreceptor en NDR de suicidas deprimidos podría representar una respuesta homeostática a una menor liberación de serotonina, con un incremento de la descarga de las neuronas serotoninérgicas. La mayor unión al autorreceptor en el NDR rostral podría contribuir a una liberación deficiente de serotonina en la corteza prefrontal ventromedial mediante una menor descarga neuronal (AU)

Serotonergic dysfunction is present in mood disorders and suicide. Brainstem 5-HT1A somatodendritic autoreceptors regulate serotonin neuron firing but studies of autoreceptor binding in the dorsal raphe nucleus (DRN) in depressed suicides report conflicting results. We sought to determine: (1) the anatomical distribution of 5-HT1A receptor binding in the DRN in depressed suicides and psychiatrically normal controls; and (2) whether sex differences in 5-HT1A binding in the DRN contribute to differences between depressed suicides and controls. Previously collected quantitative receptor autoradiograms of [[3H]8-hydroxy-2-(di-n-propyl)aminotetralin (3H-8-OH-DPAT) in postmortem tissue sections containing the DRN from drug-free suicide victims (n=10) and matched controls (n=10) were analyzed. Less total receptor binding (fmol/mg tissue·mm3) was observed in the entire DRN in depressed suicides compared with controls (p<0.05). Group differences along the rostrocaudal extent of the DRN were observed for cross-sectional 5-HT1A binding (fmol/mg tissue) and receptor binding (fmol/mg·mm3, p<0.05). Cross-sectional 5-HT1A DRN binding in depressed suicides compared with controls was higher rostrally and lower caudally. The differences between depressed suicides and controls were present in males and females, although females had more binding than males. Less autoreceptor binding in the DRN of depressed suicides may represent a homeostatic response to less serotonin release, increasing serotonin neuron firing. More autoreceptor binding in rostral DRN might contribute to deficient serotonin release in ventromedial prefrontal cortex by lower neuronal firing (AU)

Contractile effects of 5-hydroxytryptamine (5-HT) in the equine jejunum circular muscle: functional and immunohistochemical identification of a 5-HT1A-like receptor.

REASONS FOR PERFORMING STUDY: Prokinetic drugs used to treat gastrointestinal ileus in man have equivocal results in horses. In man, prokinetic drugs have 5-hydroxytryptamine4(5-HT4) receptors as their target, but little is known about the 5-HT-receptor subtypes in the equine small intestine. OBJECTIVE: Functional and immunohistochemical identification of the serotonin receptor subtype(s) responsible for the 5-HT induced contractile response in the equine circular jejunum. METHODS: Isometric organ-bath recordings were carried out to assess spontaneous and drug-evoked contractile activity of equine circular jejunum. Histological investigations by immunofluorescence analyses were performed to check for presence and localisation of this functionally identified 5-HT receptor subtype. RESULTS: Tonic contractions were induced by 5-HT in horse jejunal circular muscle. Tetrodotoxin, atropine and NG-nitro L-arginine did not modify this response. A set of 5-HT receptor subtype selective antagonists excluded interaction with 5-HT1B, 1D, 2A, 3, 4 and 7 receptors. The selective 5-HT1A receptor antagonists WAY 100635 and NAN 190 caused a clear rightward shift of the concentration-response curve to 5-HT. The contractile effect of 5-CT, that can interact with 5-HT1A, 1B, 1D, 5 and 7 receptors was also antagonised by WAY 100635, identifying the targeted 5-HT receptor as a 5-HT1A-like receptor. Immunohistology performed with rabbit polyclonal anti-5-HT1A receptor antibodies confirmed the presence of muscular 5-HT1A receptors in the muscularis mucosae, and both longitudinal and circular smooth muscle layers of the equine jejunum. CONCLUSIONS: Contractile responses in equine jejunal circular smooth muscle induced by 5-HT involves 5-HT1A-like receptors.

Solubilization of serotonin1A receptors heterologously expressed in Chinese hamster ovary cells.

1. The serotonin1A (5-HT1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. 2. We report here, for the first time, the solubilization of 5-HT1A receptors stably expressed in Chinese Hamster Ovary (CHO) cells using the zwitterionic detergent CHAPS in presence of NaCl followed by polyethylene glycol (PEG) precipitation. We show by ligand-binding assay that the 5-HT1A receptor solubilized this way is functionally active. We have optimized the efficiency of solubilization with respect to total protein and NaCl concentration. 3. Our results show that careful control of salt and protein concentration is crucial in optimal solubilization of membrane receptors heterologously expressed in cells in culture. The effective solubilization of important neurotransmitter receptors such as 5-HT1A receptors which are present in very low amounts in the native tissue may represent an important step in characterizing membrane receptors expressed in mammalian cells in culture.