Discovery of novel inhibitors of vascular endothelial growth factor-A–Neuropilin-1 interaction by structure-based virtual screening

Neuropilin-1 (NRP-1), one of the most important co-receptors of vascular endothelial growth factor-A (VEGF-A), increases its angiogenic action in several chronic diseases including cancer by increasing the activity of associated tyrosine kinase receptors, VEGFR1 and VEGFR2. Binding of VEGF-A to NRP-1 plays a critical role in pathological angiogenesis and tumor progression. Today, targeting this interaction is a validated approach to fight against angiogenesis-dependent diseases. Only anti-NRP-1 antibodies, peptide and peptidomimetic drug-candidates or hits have been developed thus far. In order to identify potent orally active small organic molecules various experimental and in silico approaches can be used. Here we report, novel promising small drug-like molecules disrupting the binding of VEGF-A₁₆₅ to NRP-1. We carried out structure-based virtual screening experiments using the ChemBridge compound collection on the VEGF-A₁₆₅ binding pocket of NRP-1. After docking and two rounds of similarity search computations, we identified 4 compounds that inhibit the biotinylated VEGF-A₁₆₅ binding to recombinant NRP-1 with K_i of about 10 μM. These compounds contain a common chlorobenzyloxy alkyloxy halogenobenzyl amine scaffold that can serve as a base for further development of new NRP-1 inhibitors.     

Detection of VEGF-Axxxb Isoforms in Human Tissues

Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A₁₆₅a, and anti-angiogenic isoforms typified by VEGF-A₁₆₅b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A₁₆₅b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A₁₆₅b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A₁₆₅b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF₁₆₅b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A₁₆₅b antibody. These findings support the conclusion that more information on the biology of VEGF-A₁₆₅b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.     

VEGF-A165 Potently Induces Human Blood–Nerve Barrier Endothelial Cell Proliferation, Angiogenesis, and Wound Healing In Vitro

Several mitogens such as vascular endothelial growth factor (VEGF) have been implicated in mammalian vascular proliferation and repair. However, the molecular mediators of human blood-nerve barrier (BNB) development and specialization are unknown. Primary human endoneurial endothelial cells (pHEndECs) were expanded in vitro and specific mitogen receptors detected by western blot. pHEndECs were cultured with basal medium containing different mitogen concentrations with or without heparin. Non-radioactive cell proliferation, Matrigel^?-induced angiogenesis and sterile micropipette injury wound healing assays were performed. Proliferation rates, number and total length of induced microvessels, and rate of endothelial cell monolayer wound healing were determined and compared to basal conditions. VEGF-A₁₆₅ in the presence of heparin, was the most potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. 1.31 nM VEGF-A₁₆₅ induced ~110 % increase in cell proliferation relative to basal conditions (～51 % without heparin). 2.62 pM VEGF-A₁₆₅ induced a three-fold increase in mean number of microvessels and 3.9-fold increase in total capillary length/field relative to basal conditions. In addition, 0.26 nM VEGF-A₁₆₅ induced ～1.3-fold increased average rate of endothelial wound healing 4–18 h after endothelial monolayer injury, mediated by increased cell migration. VEGF-A₁₆₅ was the only mitogen capable of complete wound closure, occurring within 30 h following injury via increased cell proliferation. This study demonstrates that VEGF-A₁₆₅, in the presence of heparin, is a potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. VEGF-A₁₆₅ may be an important mitogen necessary for human BNB development and recovery in response to peripheral nerve injury.     

Synthesis and structure–activity relationship of non-peptidic antagonists of neuropilin-1 receptor

Neuropilins (NRPs) are VEGF-A₁₆₅ co-receptors over-expressed in tumor cells, and considered as targets in angiogenic-related pathologies. We previously identified compound 1, the first non-peptidic antagonist of the VEGF-A₁₆₅/NRP binding, which exhibits in vivo anti-angiogenic and anti-tumor activities. We report here the synthesis and biological evaluations of new antagonists structurally-related to compound 1. Among these molecules, 4a, 4c and 4d show cytotoxic effects on HUVEC and MDA-MB-31 cells, and antagonize VEGF-A₁₆₅/NRP-1 binding. This study confirmed our key structure–activity relationships hypothesis and paved the way to compound 1 ‘hit to lead’ optimization.     

Neuropilin-1 regulates a new VEGF-induced gene, Phactr-1, which controls tubulogenesis and modulates lamellipodial dynamics in human endothelial cells

Recently, we identified a new Vascular Endothelial Growth Factor (VEGF)-A₁₆₅-induced gene Phactr-1, (Phosphatase Actin Regulator-1). We reported that Phactr-1 gene silencing inhibited tube formation in human umbilical endothelial cells (HUVECs) indicating a key role for Phactr-1 in tubulogenesis in vitro. In this study, we investigated the role of Phactr-1 in several cellular processes related to angiogenesis. We found that neuropilin-1 (NRP-1) and VEGF-R1 depletion inhibited Phactr-1 mRNA expression while NRP-2 and VEGF-R2 depletion had no effect. We described a new interaction site of VEGF-A₁₆₅ to VEGF-R1 in peptides encoded by exons 7 and 8 of VEGF-A₁₆₅. The specific inhibition of VEGF-A₁₆₅ binding on NRP-1 and VEGF-R1 by ERTCRC and CDKPRR peptides decreased the Phactr-1 mRNA levels in HUVECs indicating that VEGF-A₁₆₅-dependent regulation of Phactr-1 expression required both NRP-1 and VEGF-R1 receptors. In addition, upon VEGFA₁₆₅-stimulation Phactr-1 promotes formation and maintenance of cellular tubes through NRP-1 and VEGFR1. Phactr-1 was previously identified as protein phosphatase 1 (PP1) α-interacting protein that possesses actin-binding domains. We showed that Phactr-1 depletion decreased PP1 activity, disrupted the fine-tuning of actin polymerization and impaired lamellipodial dynamics. Taken together our results strongly suggest that Phactr-1 is a key component in the angiogenic process.     

Selection of DNA aptamers against VEGF<Subscript>165</Subscript> using a protein competitor and the aptamer blotting method

Two DNA aptamers against a tumor marker protein, human vascular endothelial growth factor (VEGF₁₆₅) were identified. In the screening process, another protein was used as the competitor to isolate those aptamers that have high specificity for the target. In addition, we evaluated the affinities of the enriched library by means of aptamer blotting. The isolated aptamers bound to VEGF₁₆₅ with a K _d value in the range of a few hundred nanomoles, and did not bind to the competitor. This selection method enabled us to efficiently select the specific aptamers against the target protein. These specific aptamers would be useful sensor elements for cancer diagnosis.     

Perlecan regulates developmental angiogenesis by modulating the VEGF-VEGFR2 axis

Using the zebrafish, we previously identified a central function for perlecan during angiogenic blood vessel development. Here, we explored the nature of perlecan function during developmental angiogenesis. A close examination of individual endothelial cell behavior revealed that perlecan is required for proper endothelial cell migration and proliferation. Because these events are largely mediated by VEGF-VEGFR2 signaling, we investigated the relationship between perlecan and the VEGF pathway. We discovered that perlecan knockdown caused an abnormal increase and redistribution of total VEGF-A protein suggesting that perlecan is required for the appropriate localization of VEGF-A. Importantly, we linked perlecan function to the VEGF pathway by efficiently rescuing the perlecan morphant phenotype by microinjecting VEGF-A₁₆₅ protein or mRNA. Combining the strategic localization of perlecan throughout the vascular basement membrane along with its growth factor-binding ability, we hypothesized a major role for perlecan during the establishment of the VEGF gradient which provides the instructive cues to endothelial cells during angiogenesis. In support of this hypothesis we demonstrated that human perlecan bound in a heparan sulfate-dependent fashion to VEGF-A₁₆₅. Moreover, perlecan enhanced VEGF mediated VEGFR2 activation of human endothelial cells. Collectively, our results indicate that perlecan coordinates developmental angiogenesis through modulation of VEGF-VEGFR2 signaling events. The identification of angiogenic factors, such as perlecan, and their role in vertebrate development will not only enhance overall understanding of the molecular basis of angiogenesis, but may also provide new insight into angiogenesis-based therapeutic approaches.     

Urea moiety as amide bond mimetic in peptide-like inhibitors of VEGF-A165/NRP-1 complex

NRP-1 is an important co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2). Many reports suggested that NRP-1 might also serve as a separate receptor for VEGF-A₁₆₅ causing stimulation of tumour growth and metastasis. Therefore, compounds interfering with VEGF-A₁₆₅/NRP-1 complex triggered interest in the design of new molecules, including peptides, as anti-angiogenic and anti-tumour drugs. Here, we report the synthesis, affinity and stability evaluation of the urea-peptide hybrids, based on general Lys(hArg)-AA²-AA³-Arg sequence, where hArg residue was substituted by Arg urea unit. Such substitution does not substantially affected affinity of compounds for NRP-1 but significantly increased their proteolytic stability in plasma.     

Depletion of the novel protein PHACTR-1 from human endothelial cells abolishes tube formation and induces cell death receptor apoptosis

Using suppression subtractive hybridisation (SSH), we identified a hitherto unreported gene PHACTR-1 (Phosphatase Actin Regulating Protein-1) in Human Umbilical Vascular Endothelial Cells (HUVECs). PHACTR-1 is an actin and protein phosphatase 1 (PP1) binding protein which is reported to be highly expressed in brain and which controls PP1 activity and F-actin remodelling. We have also reported that its expression is dependent of Vascular Endothelial Growth Factor (VEGF-A₁₆₅). To study its function in endothelial cells, we used a siRNA strategy against PHACTR-1. PHACTR-1 siRNA-treated HUVECs showed a major impairment of tube formation and stabilisation. PHACTR-1 depletion triggered apoptosis through death receptors DR4, DR5 and FAS, which was reversed using death receptor siRNAs or with death receptor-dependent caspase-8 siRNA. Our findings suggest that PHACTR-1 is likely to be a key regulator of endothelial cell function properties. Because of its central role in the control of tube formation and endothelial cell survival, PHACTR-1 may represent a new target for the development of anti-angiogenic therapy.     

Effects of elevated atmospheric CO<Subscript>2</Subscript> on the nutritional ecology of C<Subscript>3</Subscript> and C<Subscript>4</Subscript> grass-feeding caterpillars

It is plausible that the nutritional quality of C₃ plants will decline more under elevated atmospheric CO₂ than will the nutritional quality of C₄ plants, causing herbivorous insects to increase their feeding on C₃ plants relative to C₄ plants. We tested this hypothesis with a C₃ and C₄ grass and two caterpillar species with different diet breadths. Lolium multiflorum (C₃) and Bouteloua curtipendula (C₄) were grown in outdoor open top chambers at ambient (370 ppm) or elevated (740 ppm) CO₂. Bioassays compared the performance and digestive efficiencies of Pseudaletia unipuncta (a grass-specialist noctuid) and Spodoptera frugiperda (a generalist noctuid). As expected, the nutritional quality of L. multiflorum changed to a greater extent than did that of B. curtipendula when grown in elevated CO₂; levels of protein (considered growth limiting) declined in the C₃ grass, while levels of carbohydrates (sugar, starch and fructan) increased. However, neither insect species increased its feeding rate on the C₃ grass to compensate for its lower nutritional quality when grown in an elevated CO₂ atmosphere. Consumption rates of P. unipuncta and S. frugiperda were higher on the C₃ grass than the C₄ grass, the opposite of the result expected for a compensatory response to the lower nutritional quality of the C₄ grass. Although our results do not support the hypothesis that grass-specialist insects compensate for lower nutritional quality by increasing their consumption rates more than do generalist insects, the performance of the specialist was greater than that of the generalist on each grass species and at both CO₂ levels. Mechanisms other than compensatory feeding, such as increased nutrient assimilation efficiency, appear to determine the relative performance of these herbivores. Our results also provide further evidence against the hypothesis that C₄ grasses would be avoided by insect herbivores because a large fraction of their nutrients is unavailable to herbivores. Instead, our results are consistent with the hypothesis that C₄ grasses are poorer host plants primarily because of their lower nutrient levels, higher fiber levels, and greater toughness.     

Effect of long-term drought stress on leaf gas exchange and fluorescence parameters in C<Subscript>3</Subscript> and C<Subscript>4</Subscript> plants

A field study was performed on triticale, field bean, maize and amaranth, to find differences between studied species in physiological alterations resulting from progressive response as injuries and/or acclimation to long-term soil drought during various stages of plant development. The measurements of leaf water potential, electrolyte leakage, chlorophyll a fluorescence, leaf gas exchange and yield analysis were done. A special emphasis was given to the measurements of the blue, green, red and far-red fluorescence. Beside, different ratios of the four fluorescence bands (red/far-red: F ₆₉₀/F ₇₄₀, blue/red: F ₄₄₀/F ₆₉₀, blue/far-red: F ₄₄₀/F ₇₄₀ and blue/green: F ₄₄₀/F ₅₂₀) were calculated. Based on both yield analysis and measurements of physiological processes it can be suggested that field bean and maize responded with better tolerance to the water deficit in soil due to the activation of photoprotective mechanism probably connected with synthesis of the phenolic compounds, which can play a role of photoprotectors in different stages of plant development. The photosynthetic apparatus of those two species scattered the excess of excitation energy more effectively, partially through its transfer to PS I. In this way, plants avoided irreversible and/or deep injuries to PS II. The observed changes in the red fluorescence emission and in the F _v/F _m for triticale and amaranth could have occurred due to serious and irreversible photoinhibitory injuries. Probably, field bean and maize acclimatized more effectively to soil drought through the development of effective mechanisms for utilising excitation energy in the photosynthetic conversion of light accompanied by the mechanism protecting the photosynthetic apparatus against the excess of this energy.     

Manipulating the Length of the <Emphasis Type="Italic">b</Emphasis> Subunit F<Subscript>1</Subscript> Binding Domain in F<Subscript>1</Subscript>F<Subscript>0</Subscript> ATP Synthase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The peripheral stalk of F₁F₀ ATP synthase is composed of a parallel homodimer of b subunits that extends across the cytoplasmic membrane in F₀ to the top of the F₁ sector. The stalk serves as the stator necessary for holding F₁ against movement of the rotor. A series of insertions and deletions have been engineered into the hydrophilic domain that interacts with F₁. Only the hydrophobic segment from {val-121} to {ala-132} and the extreme carboxyl terminus proved to be highly sensitive to mutation. Deletions in either site apparently abolished enzyme function as a result of defects is assembly of the F₁F₀ complex. Other mutations manipulating the length of the sequence between these two areas had only limited effects on enzyme function. Expression of a b subunit with insertions with as few as two amino acids into the hydrophobic segment also resulted in loss of F₁F₀ ATP synthase. However, a fully defective b subunit with seven additional amino acids could be stabilized in a heterodimeric peripheral stalk within a functional F₁F₀ complex by a normal b subunit.     

Identifying non-invasible habitats for marine copepods using temperature-dependent <Emphasis Type="Italic">R</Emphasis><Subscript>0</Subscript>

If a non-indigenous species is to thrive and become invasive it must first persist under its new set of environmental conditions. Net reproductive rate (R ₀) represents the average number of female offspring produced by a female over its lifetime, and has been used as a metric of population persistence. We modeled R ₀ as a function of ambient water temperature (T) for the invasive marine calanoid copepod Pseudodiaptomus marinus, which is introduced to west coast of North America from East Asia by ship ballast water. The model was based on temperature-dependent stage-structured population dynamics given by a system of ordinary differential equations. We proposed a methodology to identify habitats that are non-invasible for P. marinus using the threshold of R ₀(T) < 1 in order to identify potentially invasible habitats. We parameterized the model using published data on P. marinus and applied R ₀(T) to identify the range of non-invasible habitats in a global scale based on sea surface temperature data. The model predictions matched the field evidence of species occurrences well.     

High Bcl-2/Bax ratio in Walker tumor cells protects mitochondria but does not prevent H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced apoptosis via calcineurin pathways

It has been previously shown that Walker 256 tumor cells express a high content of the anti-apoptotic protein Bcl-2 which protects mitochondria against the damaging effects of Ca²⁺. In the present study, we analyze H₂O₂-induced apoptotic death in two different types of tumor cells: Walker 256 and SCC-25. Treatment with H₂O₂ (4mM) increased reactive oxygen species generation and the concentration of cytosolic free Ca²⁺. These alterations preceded apoptosis in both cell lines. In Walker cells, which show a high Bcl-2/Bax ratio, apoptosis was dependent on calcineurin activation and independent of changes in mitochondrial membrane potential (Δ < eqid1 > _m), as well as cytochrome c release. In contrast, in SCC-25 cells, which show a lower Bcl-2/Bax ratio, apoptosis was preceded by a decrease in Δ < eqid2 > _m, mitochondrial permeability transition, and cytochrome c release. Caspase-3 activation occurred in both cell lines. The data suggest that although the high Bcl-2/Bax ratio protected the mitochondria of Walker cells from oxidative stress, it was not sufficient to prevent apoptosis through calcineurin pathways.     

Partitioning of photosynthetic electron flow between CO<Subscript>2</Subscript> assimilation and O<Subscript>2</Subscript> reduction in sunflower plants under water deficit

In sunflower (Helianthus annuus L.) grown under controlled conditions and subjected to drought by withholding watering, net photosynthetic rate (P _N) and stomatal conductance (g _s) of attached leaves decreased as leaf water potential (Ψ_w) declined from −0.3 to −2.9 MPa. Although g _s decreased over the whole range of Ψ_w, nearly constant values in the intercellular CO₂ concentrations (C _i) were observed as Ψ_w decreased to −1.8 MPa, but C _i increased as Ψ_w decreased further. Relative quantum yield, photochemical quenching, and the apparent quantum yield of photosynthesis decreased with water deficit, whereas non-photochemical quenching (q_NP) increased progressively. A highly significant negative relationship between q_NP and ATP content was observed. Water deficit did not alter the pyridine nucleotide concentration but decreased ATP content suggesting metabolic impairment. At a photon flux density of 550 μmol m⁻² s⁻¹, the allocation of electrons from photosystem (PS) 2 to O₂ reduction was increased by 51 %, while the allocation to CO₂ assimilation was diminished by 32 %, as Ψ_w declined from −0.3 to −2.9 MPa. A significant linear relationship between mean P _N and the rate of total linear electron transport was observed in well watered plants, the correlation becoming curvilinear when water deficit increased. The maximum quantum yield of PS2 was not affected by water deficit, whereas q_P declined only at very severe stress and the excess photon energy was dissipated by increasing q_NP indicating that a greater proportion of the energy was thermally dissipated. This accounted for the apparent down-regulation of PS2 and supported the protective role of q_NP against photoinhibition in sunflower.     

Differential actions of VEGF-A isoforms on perichondrial angiogenesis during endochondral bone formation

During endochondral bone formation, vascular invasion initiates the replacement of avascular cartilage by bone. We demonstrate herein that the cartilage-specific overexpression of VEGF-A₁₆₄ in mice results in the hypervascularization of soft connective tissues away from cartilage. Unexpectedly, perichondrial tissue remained avascular in addition to cartilage. Hypervascularization of tissues similarly occurred when various VEGF-A isoforms were overexpressed in the chick forelimb, but also in this case perichondrial tissue and cartilage were completely devoid of vasculature. However, following bony collar formation, anti-angiogenic properties in perichondrial tissue were lost and perichondrial angiogenesis was accelerated by VEGF-A₁₄₆, VEGF-A₁₆₆, or VEGF-A₁₉₀. Once the perichondrium was vascularized, osteoclast precursors were recruited from the circulation and the induction of MMP9 and MMP13 can be observed in parallel with the activation of TGF-β signaling. Neither perichondrial angiogenesis nor the subsequent cartilage vascularization was found to be accelerated by the non-heparin-binding VEGF-A₁₂₂ or by the VEGF-A₁₆₆ΔE₁₆₂-R₁₆₆ mutant lacking a neuropilin-binding motif. Hence, perichondrial angiogenesis is a prerequisite for subsequent cartilage vascularization and is differentially regulated by VEGF-A isoforms.     

Evaluation of the antibacterial activity and treatment of acne with SiO<Subscript>2</Subscript>-ionized loess

SiO₂-ionized loess was prepared from the reaction of loess and sodium hydroxide at 1,400°C for 2 h. The antibacterial activity of SiO₂-ionized loess against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aerusinosa, and Propionobacterium acnes causing acne was examined by comparing the results against those of untreated loess, and treatment efficacy was examined for treating acne using soap containing SiO₂-ionized loess. The minimum inhibitory concentration value of SiO₂-ionized loess against S. aureus, P. aerusinosa, B. subtilis, E. coli, and P. acne was 10.0, 10.0, 2.5, 5.0, and 2.5 mg/mL, respectively. However, medium containing untreated loess had no antimicrobial activity. A treatment efficacy test revealed that acne symptoms decreased as the duration of using soap containing SiO₂-ionized loess increased.     

Molecular modeling and dynamics simulation of a histidine-tagged cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript>

Although an affinity tag such as six consecutive histidines, (His)₆-tag, has been widely used to obtain high quantity of recombinant proteins, little is known about its influences on heme proteins for lack of structural information. When (His)₆-tag was introduced to the N-terminus of a small heme protein, cytochrome b ₅, experimental results showed the resultant protein, (His)₆-cyt b ₅, has similar property and function to that of isolated cyt b ₅. To provide structural information for this observation, we herein performed a structural prediction of (His)₆-cyt b ₅ by molecular modeling in combination with molecular dynamics simulation. The predicted structure, as assessed by a series of criteria with good quality, reveals that the (His)₆-tag adopts a helical conformation and packs against the hydrophobic core 2 of cyt b ₅ through salt bridges, hydrogen bonding and hydrophobic interactions. The heme group, with the axial His ligands slightly rotated, was found to have similar conformation as in isolated cyt b ₅, which indicates that the N-terminal (His)₆-tag does not alter the heme active site, resulting in similar dynamics properties for core 1. This study provides valuable information of interactions between (His)₆-tag and the rest of the protein, aiding in rational design and application of functional His-tagged proteins.     

Membrane Organization of the Serotonin<Subscript><Emphasis Type="Bold">1A</Emphasis></Subscript> Receptor Monitored by a Detergent-Free Approach

1. Insolubility of membrane constituents in nonionic detergents such as Triton X-100 has been a widely used biochemical criterion to indicate their localization in membrane domains. However, concerns on the possibility of membrane perturbation in the presence of detergents have led to the development of detergent-free approaches. 2. We have explored the organization of the serotonin_1A receptor, an important G-protein coupled receptor, from bovine hippocampus and CHO cells using a detergent-free approach in order to address the points of agreement with our previous results using Triton X-100. 3. A significant fraction of the serotonin_1A receptor has been found to be localized in a heavy density fraction obtained using a detergent-free approach to isolate membrane domains. In addition, we have characterized the membrane fractions isolated in terms of their lipid composition and membrane physical properties. 4. The results obtained on the membrane localization of the serotonin_1A receptor from the present experiments using a detergent-free approach correlate well with our earlier findings obtained using a detergent-based method (Kalipatnapu, S., and Chattopadhyay, A., FEBS Lett. 576:455–460, 2004). These results provide important information on the membrane organization of the hippocampal serotonin_1A receptor and are relevant in view of the concerns on the use of detergent in determination of membrane organization of constituent proteins and lipids.