天花粉蛋白引起敏感细胞膜上G蛋白激活的初步研究

天花粉蛋白(Trichosanthin,TCS)是一种单链核糖体失活蛋白,具引产、抗肿瘤、抗HIV等多种生物学功能。天花粉蛋白专一性杀伤敏感细胞的机制一直未被研究清楚。本文首次以生物分子相互作用分析(BIA)证明在天花粉蛋白敏感的细胞膜上存在着能与天花粉蛋白专一结合的组分。我们进一步利用[~(35)S]GTPγS结合实验发现天花粉蛋白能够激活敏感细胞膜上的G蛋白,而对不敏感细胞没有相应的G蛋白激活。这些结果表明了在敏感细胞膜上天花粉蛋白特异受体的存在。     

毒蕈碱样乙酰胆碱受体及其信号转导

毒蕈碱样乙酰胆碱受体（MAChRs）是G蛋白偶联受体（GPCRs）超家族中的一员,具有该家族特性的结构和信号转导方式。GTP结合蛋白（Gproteins）是一类具有GTP酶活性的蛋白质,由α、β、γ三个亚基构成。其中α亚基结合GDP或GTP,分别代表G蛋白的非活化和活化状态。M受体与Gi/Go或Gq／11间的作用机制仍在探讨中,但基本过程与Gs介导的信号转导模式相似。激动剂持续作用后,G蛋白偶联受体激酶和阻滞蛋白导受体脱敏和内吞。     

趋化因子受体 CCR5 亲合短肽的筛选

趋化因子受体 5 (CCR5) 是 HIV-1 与宿主细胞结合的辅助因子之一,其功能缺失或被 CCR5 拮抗剂封闭则会阻止 HIV-1 感染细胞 . 为得到与 CCR5 特异结合的肽类拮抗剂,采用噬菌体展示技术,以稳定表达 CCR5 的 CHO 细胞 (CHO/CCR5) 作为靶标,通过噬菌体随机 12 肽库筛选与 CCR5 特异结合的多肽;经过四轮筛选后,挑选 20 个阳性噬菌体克隆进行测序,从中得到 11 个含有 AFDWTFVPSLIL 序列的小分子肽 . 含该序列的噬菌体能与抗人 CCR5 单抗 (2D7) 竞争性结合 CCR5 ,且合成肽 AFDWTFVPSLIL 对趋化因子 RANTES 与 CHO/CCR5 的结合具有明显的抑制作用,初步证明该小肽与 CCR5 具有特异性结合作用 .     

CXCR4/CXCL12 在肿瘤中的研究进展

研究表明趋化因子及其受体在胚胎发育、干细胞迁移以及各种免疫反应中发挥重要作用,是许多生理及病理过程中细胞运动的重要因素。趋化因子受体CXCR4是一个由352个氨基酸构成的、7次跨膜的G蛋白偶联受体。趋化因子CXCL12为其特异性受体。研究发现,CXCR4/CXCL12在多种肿瘤中都有表达,在肿瘤的生长、血管生成、转移等方面发挥着重要作用。与正常组织相比,肿瘤组织及转移灶CXCR4高表达。因此,对CXCR4/CXCL12轴在肿瘤病生理中的作用机制进行进一步研究,很可能为肿瘤的治疗及对肿瘤转移的预防提供一个新的思路。我们现在就对其在肿瘤病生理中的作用做一综述。     

大鼠Gs alpha亚基的原核表达和纯化

用 PCR的方法 ,在大鼠 Gs alpha亚基的 C端引入了 6个外源组氨酸 (即 6×His- Tag)并以此为纯化标记 .构成的表达载体 p QE60 /rat Gsα( L)在大肠杆菌 BL2 1 ( DE3)中获得了稳定的表达 .经 DEAE- Sephacel离子交换柱和 Ni- NTA Agarose亲和层析获得纯化的具有较高 [35S]- GTPγS结合活力的重组大鼠 Gs alpha亚基     

趋化因子CXCL1在肿瘤中的作用

趋化因子及其受体在许多生物学过程(如炎症发生、血管生成等)中起重要的作用。趋化因子CXCL1是单亚基趋药性细胞因子,该蛋白主要通过特异性结合G蛋白耦联受体CXCR2,在多种肿瘤的生长、增殖、转移和侵袭以及血管新生中起重要调控作用。该文重点阐述了趋化因子CXC亚家族成员CXCL1在肿瘤中的功能,并对其上游调控因子进行分析,深入探讨了CXCL1与肿瘤的相互关系。     

CXCR7 的生物学功能及其在肿瘤发生发展中的 作用研究进展

趋化因子受体是一类G 蛋白偶联的7 次跨膜受体,其与配体结合能参与调控多种生理和病理学过程。以往,CXCR4 一直被认为 是趋化因子CXCL12 的唯一受体。然而近年来的研究表明CXCL12 能与另一种新受体CXCR7 结合,并在调控肿瘤转移、血管生成和细 胞粘附等方面起着重要作用,CXCR7 由此成为肿瘤治疗的潜在靶点。介绍CXCR7 的结构、生物学功能以及CXCR7 在肿瘤发生发展中 的作用。     

人RANTES基因克隆及其体外表达

1995年,Cocchi等[1]发现RANTES、MIP-1α和MIP-1β等β-趋化因子具有抗HIV-1感染活性.1997年,Feng等[2]和Deng等[3]证实β-趋化因子受体CXCR4和CCR5分别是HIV-1侵染T淋巴细胞和巨噬细胞的辅助受体(co-receptor).T淋巴细胞嗜性(T-tropism)分离株被称为X4毒株,巨噬细胞嗜性(M-tropism)分离株则被称为R5毒株[4].RANTES与CCR5有着高度的亲和力,二者的结合可对HIV-1的细胞附着产生空间位阻效应,并下调CCR5在细胞表面的表达.这一结果使RANTES抗HIV-1感染机制在分子水平上得到合理的解释.最近,Garzino-Demo等[5]证明,β-趋化因子的诱导分泌与HIV-1感染后疾病进程的控制有着密切的关系,而且人群中β-趋化因子水平存在着显著的个体差异,表明β-趋化因子对艾滋病具有潜在的预防和治疗价值.为此,我们在克隆人RAN-TES基因的基础上,在体外转录与翻译系统中实现了该基因的表达,有利于今后进一步开展艾滋病的基因治疗.     

趋化因子CXCL9/Mig的研究进展

趋化因子CXCL9又称为mig,即monokine induced by IFN-γ(IFN-γ诱导的单核因子),是趋化因子CXC亚族的一员,体内主要由IFN-γ刺激的巨噬细胞和神经胶质细胞产生,体外则可由内皮细胞、粒细胞等在IFN-γ和TLR配体协同作用下产生。CXCL9的受体CXCR3为七次跨膜的G蛋白偶联受体。CXCL9对激活的T淋巴细胞和肿瘤浸润淋巴细胞具有趋化作用,但是对粒细胞和单核细胞没有作用。本文主要就CXCL9的结构与生化特征,其在免疫、移植排斥、肿瘤治疗等方面所起的作用,进行了较为系统的综述。     

天然Goα的体外棕榈酰化

从牛脑皮层膜中提纯天然Goα,并成功实现了天然Goα的体外棕榈酰化.天然Goα的体外棕榈酰化具有特异性,其修饰位点与体内棕榈酰化位点相同,发现纯化的天然Goα仍然保留较大比率的内源性棕榈酰化,而DTT的处理能大大提高表观棕榈酰化效率.棕榈酰化对天然Goα结合GTPγS的性质并无明显影响,但能影响其对大单层脂质体的亲和性.天然Goα的体外棕榈酰化模型的建立为直接研究棕榈酰化在G蛋白信号转导中的功能提供了良好基础.     

Signal amplification in HL-60 granulocytes. Evidence that the chemotactic peptide receptor catalytically activates guanine-nucleotide-binding regulatory proteins in native plasma membranes

Receptors for the chemotactic peptide fMet-Leu-Phe (fMet, N-formylmethionine) are present in membranes of myeloid differentiated human leukemia (HL-60) cells and stimulate phospholipase C via a pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein(s) [G-protein(s)]. We have developed methods for the assessment of formyl-peptide-receptor-stimulated binding of radiolabeled guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[S]) to native HL-60 membranes. Agonist stimulation of [35S]GTP[S] association with the membrane was minimal (less than or equal to 20%) when GTP[S] was the sole nucleotide present in the incubation medium. In contrast, receptor activation led to a marked (up to sixfold) stimulation of [35S]GTP[S] binding when GDP or GTP were present in high (greater than 100-fold) excess of [35S]GTP[S]. The increase in [35S]GTP[S] binding caused by the chemotactic agonist was strictly dependent on the presence of Mg2+ and was significantly increased by Na+. Agonist-independent binding of [35S]GTP[S] and the increase due to the chemotactic agonist were markedly attenuated by both pertussis and cholera toxin. Comparison of the number of chemotactic-peptide-sensitive [35S]GTP[S]-binding sites to the number of chemotactic peptide receptors present in HL-60 membranes provided direct evidence that a single formyl-peptide receptor is capable of catalyzing the binding of [35S]GTP[S] to, and thus the activation of, multiple (up to 20) G-proteins in native plasma membranes.     

Activation of a pertussis toxin-sensitive, inhibitory G-protein is necessary for steroid-mediated oocyte maturation in spotted seatrout

Oocyte maturation (OM) is initiated in lower vertebrates and echinoderms when maturation-inducing substances (MIS) bind oocyte membrane receptors. This study tested the hypothesis that activation of a G_i protein is necessary for MIS-mediated OM in spotted seatrout. Addition of MIS significantly decreased adenylyl cyclase activity in a steroid specific, pertussis toxin (PTX)-sensitive manner in oocyte membranes and microinjection of PTX into oocytes inhibited MIS-induced OM, suggesting the steroid activates a G_i protein. MIS significantly increased [³⁵S]GTPγS binding to ovarian membranes, confirming that MIS receptor binding activates a G-protein, and immunoprecipitation studies showed the increased [³⁵S]GTPγS binding was associated with Gα_i1-3 proteins. Radioligand binding studies in ovarian membranes using GTPγS and PTX demonstrated that the MIS binds a receptor coupled to a PTX-sensitive G-protein. This study provides the first direct evidence in a vertebrate model that MIS-induced activation of a G_i protein is necessary for OM. These results support a mechanism of MIS action involving binding to a novel, G-protein coupled receptor and activation of an inhibitory G-protein, the most comprehensive and plausible model of MIS initiation of OM proposed to date.     

Muscarinic acetylcholine receptor-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) to guanine-nucleotide-binding proteins in cardiac membranes

Receptor-regulated binding of the labeled GTP analog, guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP[S]), to guanine-nucleotide-binding proteins (G-proteins) was studied in porcine atrial membranes enriched in muscarinic acetylcholine (mACh) receptors. Binding of [35S]GTP[S] to the membranes was not or only slightly affected by the cholinergic agonist, carbachol, unless a second nucleotide was simultaneously present in the binding assay. This additional nucleotide requirement was best fulfilled by GDP, being maximally effective at 0.1-1 microM. In contrast, the GDP analog, guanosine 5'-O-(2-thiodiphosphate), could not replace GDP in promoting carbachol-induced increase in [35S]GTP[S] binding. In addition to GDP, agonist-induced stimulation of [35S]GTP[S] binding to porcine atrial membranes required the presence of Mg2+, being half-maximally and maximally effective at about 30 microM and 300 microM, respectively. Addition of NaCl, which decreased control binding measured in the presence of GDP alone, had no effect on the maximal extent of agonist-stimulated binding, but reduced the potency of carbachol in stimulating [35S]GTP[S] binding. Under optimal conditions, carbachol increased the binding of [35S]GTP[S] without apparent lag phase up to about 2.5-fold, with half-maximal and maximal increase being observed at 5-10 microM and 100 microM, respectively. The agonist-induced stimulation was competitively antagonized by the mACh receptor antagonist, atropine. The number of GTP[S] binding sites under receptor control was two--three-fold higher than the number of mACh receptors in the porcine atrial membranes used. Pretreatment of the membranes with pertussis toxin under conditions leading to 95% ADP-ribosylation of the toxin-sensitive G-protein alpha-subunits markedly reduced agonist-stimulated [35S]GTP[S] binding, with, however, about 30% stimulation still remaining. The data presented indicate that agonist-stimulated binding of [35S]GTP[S] to G-proteins can be a sensitive assay for measuring receptor-regulated G-protein activation in native membranes and, furthermore, suggest that one agonist-activated mACh receptor can activate two or three cardiac G-proteins, being mainly members of the pertussis-toxin-sensitive G-proteins.     

Modulation by Neuropeptide FF of the interaction of Mu-opioid (MOP) receptor with G-proteins

The Neuropeptide FF (NPFF) system is known to modulate the effects of opioids in vivo and in vitro. In the present study, we have investigated the effect of NPFF agonists on the coupling of the Mu-opioid (MOP) receptor to G-proteins in a model of SH-SY5Y cells transfected with NPFF₂ receptor, in which the neuronal anti-opioid activity of NPFF was previously reproduced. Activation of G-proteins was monitored by [³⁵S]GTPγS binding assay and analysis of G-protein subunits associated with MOP receptors was performed by Western blotting after immunoprecipitation of the receptor. The results demonstrate that concentrations of NPFF agonists that produce a cellular anti-opioid effect, did not affect the ability of the opioid agonist DAMGO to activate G-proteins. However, at saturating concentration of agonist or when expression of receptor was high, opioid and NPFF agonists did not stimulate [³⁵S]GTPγS binding in an additive manner, indicating that both receptors share a common fraction of a G-protein pool. In addition, stimulation of NPFF receptors in living cells modified the G-protein environment of MOP receptor by favoring its interaction with α_s, α_i2 and β subunits. This change in G-protein coupling to MOP receptor might participate in the mechanism by which NPFF agonists reduce the inhibitory activity of opioids.     

Interleukin-1 signal transduction. Increased GTP binding and hydrolysis in membranes of a murine thymoma line (EL4)

The post-receptor events which follow the binding of interleukin 1 (IL1) to cells are unclear. The present studies provide evidence for the activation of a guanine nucleotide binding protein (G protein) by IL1 in the membranes of an IL1 receptor-rich strain (NOB-1) of the EL4 murine thymoma line. IL1 alpha and beta increased the binding of the GTP analogue [35S]guanosine 5'-[gamma-thiol]trisphosphate (GTP gamma S) to membranes prepared from these cells. By 1 min after addition of IL1 there was a 2-fold enhancement in binding which was dose dependent in the range 0.1-100 ng/ml. A qualitatively similar result was obtained with IL1 beta although it was 10 times less potent. Specific neutralizing antisera to IL1 alpha and IL1 beta abolished the response. Experiments in which the concentration of [35S]GTP gamma S was varied revealed that IL1 increased the affinity of the binding sites for [35S]GTP gamma S and not their number. IL1 alpha was shown to stimulate GTPase activity in the membranes, the time and concentration dependence of this was similar to that observed for increased [35S]GTP gamma S binding. Half-maximal enhancement of [35S]GTP gamma S binding by IL1 alpha, measured after 4 min, occurred at 5% IL1 receptor occupancy. Maximal stimulation was achieved when 30% of receptors were occupied. Experiments with pertussis and cholera toxins revealed that pretreating membranes with pertussis toxin (100 ng/ml) inhibited by 50% the IL1-induced [35S]GTP gamma S binding and [gamma-32P]GTP hydrolysis. Cholera toxin (100 ng/ml) was without effect. However, both pertussis and cholera toxins at concentrations of 100 ng/ml inhibited IL1-induced IL2 secretion in EL4 NOB-1 cells. These results show that the IL1 receptor of a responsive thymoma line activates, and may be coupled to, a G protein(s). This is a possible mechanism of IL1 signal transduction.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2

To investigate the regulation of the CCR1 chemokine receptor, a rat basophilic leukemia (RBL-2H3) cell line was modified to stably express epitope-tagged receptor. These cells responded to RANTES (regulated upon activation normal T expressed and secreted), macrophage inflammatory protein-1alpha, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca(2+) mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca(2+) mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (DeltaCCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca(2+) mobilization. S2, S3, and DeltaCCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization, and were only partially desensitized by RANTES, relative to S1 and CCR1. To study CCR1 cross-regulation, RBL cells co-expressing CCR1 and receptors for interleukin-8 (CXCR1, CXCR2, or a phosphorylation-deficient mutant of CXCR2, 331T) were produced. Interleukin-8 stimulation of CXCR1 or CXCR2 cross-phosphorylated CCR1 and cross-desensitized its ability to stimulate GTPase activity and Ca(2+) mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca(2+) mobilization by S3 and DeltaCCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and DeltaCCR1, which produced sustained signals, cross-phosphorylated and cross-desensitized responses to CXCR1 as well as CXCR2. Taken together, these results indicate that CCR1-mediated responses are regulated at several steps in the signaling pathway, by receptor phosphorylation at the level of receptor/G protein coupling and by an unknown mechanism at the level of phospholipase C activation. Moreover selective cross-regulation among chemokine receptors is, in part, a consequence of the strength of signaling (i.e. greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization) which is inversely correlated with the receptor's susceptibility to phosphorylation. Since many chemokines activate multiple chemokine receptors, selective cross-regulation among such receptors may play a role in their immunomodulation.     

Investigation of non-CB1, non-CB2 WIN55212-2-sensitive G-protein-coupled receptors in the brains of mammals,birds, and amphibians

Abstract

Purpose: Previous studies have found non-CB₁ non-CB₂ G-protein-coupled receptors in rodents that are activated by the aminoalkylindole cannabinoid agonist WIN55212-2. This work obtained evidence for the presence or absence of similar receptors in the brains of other mammals, birds and amphibians.

Materials and methods: Antagonism of the stimulation of [³⁵S]GTPγS binding by WIN55212-2 and CP55940 was assessed in multiple CNS regions of rat and canine, and in whole brain membranes from shrew, pigeon, frog and newt. A bioinformatics approach searched for orthologs of GRP3, GPR6, and GPR12 (closely related to cannabinoid receptors) in the genomes of these or related species. Orthologs were examined for amino acid motifs known to impart functionality to receptors.

Results: In mammals and pigeon, but not amphibians, a significant fraction of the stimulation of [³⁵S]GTPγS binding by WIN55212-2 was not blocked by the CB₁ antagonist SR141716A. BLAST searches found that GPR3 was restricted to mammals. GPR12 orthologs existed in all species, and they shared identical amino acid motifs. GPR6 orthologs existed all species, but with significant departures in the identity of some critical amino acids in bird, more so in amphibian.

Conclusions: The portion of WIN55212-2-stimulated [³⁵S]GTPγS binding that was antagonized by SR141716A was consistent with stimulation via CB₁ receptors, indicating that antagonist-insensitive activity was via a different G-protein coupled receptor. Pharmacological evidence of this receptor was found in the brains of mammals and pigeon, but not frog or newt. Bioinfomatics results implicate GPR6 as a possible candidate for the additional WIN55212-2-sensitive receptor.     

Activation of G-proteins in the mouse pons/medulla by beta-endorphin is mediated by the stimulation of mu- and putative epsilon-receptors

The activation of mu-, delta- and kappa1-opioid receptors by their respective agonists increases the binding of the non-hydrolyzable GTP analog guanosine-5'-(gamma-thio)-triphosphate (GTPgammaS) to G proteins. Beta-endorphin is an endogenous opioid peptide which binds nonselectively to mu-, delta- and putative epsilon-opioid receptors. The present experiment was designed to determine which opioid receptors are involved in the stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the mouse pons/medulla. The mouse pons/medulla membranes were incubated in an assay buffer containing 50 pM [35S]GTPgammaS, 30 microM GDP and various concentrations of beta-endorphin. Beta-endorphin (0.1 nM-10 microM) increased [35S]GTPgammaS binding in a concentration-dependent manner, and 10 microM beta-endorphin produced a maximal stimulation of approximately 260% over baseline. This stimulation of [35S]GTPgammaS binding by beta-endorphin was partially attenuated by the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA), but not by the delta-opioid receptor antagonist naltrindole (NTI) or the kappa1-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Beta-endorphin stimulated [35S]GTPgammaS binding by about 80% in the presence of 10 microM beta-FNA, 30 nM NTI and 100 nM nor-BNI. The same concentrations of these antagonists completely blocked the stimulation of [35S]GTPgammaS binding induced by 10 microM [D-Ala2,NHPhe4,Gly-ol]enkephalin, [D-Pen(2,5)]enkephalin and U50,488H, respectively. Moreover, the residual stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the presence of the three opioid receptor antagonists was significantly attenuated by 100 nM of the putative epsilon-opioid receptor partial agonist beta-endorphin (1-27). These results indicate that the stimulation of [35S]GTPgammaS binding induced by beta-endorphin is mediated by the stimulation of both mu- and putative epsilon-opioid receptors in the mouse pons/medulla.     

Guanosine 5′-(γ-[35S]Thio)triphosphate Autoradiography Allows Selective Detection of Histamine H3 Receptor-Dependent G Protein Activation in Rat Brain Tissue Sections

Abstract: Histamine elicits its biological effects via three distinct G protein-coupled receptors, termed H₁, H₂, and H₃. We have used guanosine 5′-(γ-[³⁵S]thio)triphosphate (GTPγ[³⁵S]) autoradiography to localize histamine receptor-dependent G protein activation in rat brain tissue sections. Initial studies revealed that in basal conditions, adenosine was present in tissue sections in sufficient concentrations to generate an adenosine A₁ receptor-dependent GTPγ[³⁵S] signal in several brain regions. All further incubations therefore contained 8-cyclopentyl-1,3-dipropylxanthine (10 µM), a selective A₁ receptor antagonist. Histamine elicited dose-dependent increments in GTPγ[³⁵S] binding to discrete anatomical structures, most notably the caudate putamen, cerebral cortex, and substantia nigra. The overall anatomical pattern of the histamine-evoked binding response closely reflects the known distribution of H₃ binding sites and was faithfully mimicked by N^α-methylhistamine, (R)-α-methylhistamine, and immepip, three H₃-selective agonists. In all regions examined, the GTPγ[³⁵S] signal was reversed with thioperamide and clobenpropit, two potent H₃-selective antagonists, whereas mepyramine, a specific H₁ antagonist, and cimetidine, a prototypic H₂ antagonist, proved ineffective. These data indicate that in rat brain tissue sections, GTPγ[³⁵S] autoradiography selectively detects H₃ receptor-dependent signaling in response to histamine stimulation. As the existing evidence suggests that GTPγ[³⁵S] autoradiography preferentially reveals responses to G_i/o-coupled receptors, our data indicate that most, if not all, central H₃ binding sites represent functional receptors coupling to G_i/o, the inhibitory class of G proteins. Besides allowing more detailed studies on H₃ receptor signaling within anatomically restricted regions of the CNS, GTPγ[³⁵S] autoradiography offers a novel approach for functional in vitro screening of H₃ ligands.