Demonstration of the transmembrane nature of the acetylcholine receptor by labeling with anti-receptor antibodies.

Antibodies raised in rabbits to Triton-solubilized, purified acetylcholine receptor from Torpedo californica were used to immunospecifically label intact T. californica electroplaque membrane vesicles attached to cover slips and oriented with the extracellular face of the synaptic membrane facing outward. Hemocyanin conjugated to Protein A was then used as a marker, making the antibody binding visible at the electron microscope level. Parallel labeling experiments were performed on vesicles attached to cover slips and sheared by sonication, leaving their cytoplasmic faces fully exposed to the labeling solution. While differences in antibody populations among different rabbits were observed, antigenic determinants of the receptor were present on both faces of the membrane, with those on the extracellular side more numerous than those on the cytoplasmic side, demonstrating the transmembrane nature of the receptor molecule.

Acetylcholine receptor: complex of homologous subunits.

The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

Diet-induced obese mice develop peripheral, but not central, resistance to leptin.

Leptin administration reduces obesity in leptin-deficient ob/ob mice; its effects in obese humans, who have high circulating leptin levels, remain to be determined. This longitudinal study was designed to determine whether diet-induced obesity in mice produces resistance to peripheral and/or central leptin treatment. Obesity was induced in two strains of mice by exposure to a 45% fat diet. Serum leptin increased in proportion to body weight (P < 0.00001). Whereas C57BL/6 mice initially responded to peripherally administered leptin with a marked decrease in food intake, leptin resistance developed after 16 d on high fat diet; mice on 10% fat diet retained leptin sensitivity. In AKR mice, peripheral leptin significantly decreased food intake in both 10 and 45% fat-fed mice after 16 d of dietary treatment. However, after 56 d, both groups became resistant to peripherally administered leptin. Central administration of leptin to peripherally leptin-resistant AKR mice on 45% fat diet resulted in a robust response to leptin, with a dose-dependent decrease in food intake (P < 0.00001) and body weight (P < 0.0001) after a single intracerebroventricular infusion. These data demonstrate that, in a diet-induced obesity model, mice exhibit resistance to peripherally administered leptin, while retaining sensitivity to centrally administered leptin.

A selective human beta3 adrenergic receptor agonist increases metabolic rate in rhesus monkeys.

Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.

(Thr-59)-insulin-like growth factor I stimulates 2-deoxyglucose transport in BC3H1 myocytes through the insulin-like growth factor receptor, not the insulin receptor.

The murine non-fusing muscle cell line contains distinct receptors for insulin and insulin-like growth factors. Pretreatment of myocytes with insulin for 20 h at 37 degrees C inhibits the binding of [125I]iodoinsulin by 60% without affecting the binding of [125I]iodoinsulin-like growth factor I. The ED50 values for down-regulation of the insulin and insulin-like growth factor receptor by their respective ligands are 1 nM and 3 nM, respectively. Insulin, (Thr-59)-insulin-like growth factor I and multiplication-stimulating activity stimulate 2-[3H]deoxyglucose transport in myocytes with ED50 values of 5 nM, 5.6 nM and 33 nM, respectively. In order to determine whether (Thr-59)-insulin-like growth factor I stimulates 2-[3H]deoxyglucose transport in myocytes via its own receptor or the insulin receptor, we determined the activity of these peptides after down-regulation of the insulin receptor. The rate of 2-[3H]deoxyglucose transport in myocytes pretreated with insulin (5 nM) is elevated but returns to control levels by 1 h after the washout of insulin. The dose-response curve for insulin-stimulated 2-[3H]deoxyglucose transport is shifted to the right (ED50 greater than 100 nM) immediately after insulin washout but is normal by 1 h after insulin washout. In contrast, the dose-response curve for (Thr-59)-insulin-like growth factor I is unchanged in insulin-pretreated cells immediately after insulin washout. These data show that (Thr-59)-insulin-like growth factor I stimulates 2-[3H]deoxyglucose transport in myocytes by acting through an insulin-like growth factor receptor and not through the insulin receptor. Since multiplication-stimulating activity is 6-fold less active than (Thr-59)-insulin-like growth factor, they both may be acting through a type 1 insulin-like growth factor receptor.

Genetic approaches to the determination of structure-function relationships of G protein-coupled receptors.

The beta-adrenergic receptor (beta AR), which has been extensively characterized pharmacologically, serves as a useful model system for the analysis of the structure-function relationships of G protein-coupled receptors. Genetic and biochemical analysis has revealed that the ligand binding domain of the receptor involves residues within the hydrophobic transmembrane core of the protein. Molecular substitution experiments suggest that adrenergic agonists and antagonists are anchored to the receptor through an ionic interaction between Asp113 in the third hydrophobic region of the receptor and the protonated amine group of the ligand. In addition, catecholamine agonists are bound through hydrogen bonding interactions between two serine residues in the fifth hydrophobic domain of the receptor and the catechol hydroxyl groups of the ligand. Agonist-mediated activation of the G protein Gs requires residues within the cytoplasmic loop linking the fifth and sixth transmembrane helices which are predicted to form amphipathic alpha-helices. The strong structural similarities among G protein-coupled receptors imply that the information gained from genetic analysis of the beta AR should be applicable to other hormone and neurotransmitter receptors of this class.

Molecular approaches to the discovery of new treatments for obesity.

The treatment of obesity requires modulation of both energy intake and energy expenditure, and pharmaceutical treatments are being developed to complement the traditional means of dietary restriction and exercise. The recent discovery of the protein leptin, which modulates both food intake and energy expenditure, has provided a new tool with which to define and analyze potential pathways for pharmacological intervention. Neurotransmitters, such as neuropeptide Y (NPY) and norepinephrine, act downstream of leptin to modulate energy homeostasis. Specific subtypes of the receptors for these neurotransmitters represent promising molecular targets for the discovery of new drugs for the treatment of obesity.

Structural model of antagonist and agonist binding to the angiotensin II, AT1 subtype, G protein coupled receptor.

BACKGROUND: The family of G protein coupled receptors is the largest and perhaps most functionally diverse class of cell-surface receptors. Due to the difficulty of obtaining structural data on membrane proteins there is little information on which to base an understanding of ligand structure-activity relationships, the effects of receptor mutations and the mechanism(s) of signal transduction in this family. We therefore set out to develop a structural model for one such receptor, the human angiotensin II receptor. RESULTS: An alignment between the human angiotensin II (type 1; hAT1), human beta 2 adrenergic, human neurokinin-1, and human bradykinin receptors, all of which are G protein coupled receptors, was used to generate a three-dimensional model of the hAT1 receptor based on bacteriorhodopsin. We observed a region within the model that was congruent with the biogenic amine binding site of beta 2, and were thus able to dock a model of the hAT1 antagonist L-158,282 (MK-996) into the transmembrane region of the receptor model. The antagonist was oriented within the helical domain by recognising that the essential acid functionality of this antagonist interacts with Lys199. The structural model is consistent with much of the information on structure-activity relationships for both non-peptide and peptide ligands. CONCLUSIONS: Our model provides an explanation for the conversion of the antagonist L-158,282 (MK-996) to an agonist by the addition of an isobutyl group. It also suggests a model for domain motion during signal transduction. The approach of independently deriving three-dimensional receptor models and pharmacophore models of the ligands, then combining them, is a powerful technique which helps validate both models.

Isolation of a preadipocyte cell line from rat bone marrow and differentiation to adipocytes.

A unique population of rat adipocyte precursor cells was derived from normal rat bone marrow. The epitheloid-like preadipocytes were isolated from a mixed culture of bone marrow cells by a combination of differential trypsinization, enrichment by Ficoll gradient centrifugation, and differential seeding. This cell line, designated RBM-Ad, can be fully differentiated into multilocular adipocytes morphologically resembling brown adipose tissue. No changes in the differentiation pattern are observed during propagation of these cells, and they have been successfully carried and differentiated up to passage 49. Histological staining of differentiated cells with Sudan black, Sudan IV, and oil red O indicates the presence of lipids in intracellular vesicles. The nonselective beta-adrenergic agonist isoproterenol stimulates adenylyl cyclase activity in both preadipocytes and differentiated adipocytes. In contrast, BRL-37344, a beta 3-adrenergic receptor-specific agonist, stimulates adenylyl cyclase activity and glycerol release in differentiated adipocytes, but not preadipocytes. In addition, differentiated adipocytes contain messenger RNA encoding the brown adipose-specific protein, thermogenin. Thus, this rat preadipocyte cell line can be differentiated into adipocytes that histologically and functionally resemble brown adipose tissue.

Pharmacological characterization of a recently described human beta 3-adrenergic receptor mutant.

The beta 3-adrenergic receptor is the predominant subtype of beta-adrenergic receptor expressed in adipose tissue. Recently, a naturally occurring mutation in the human beta 3-receptor gene has been described which results in substitution of the tryptophan residue at position 64 in the first intracellular loop with an arginine residue. The polymorphism, which is prevalent in the human population, has been associated with increases in some parameters of obesity and Type II diabetes. In order to characterize the pharmacological effects of this amino acid substitution, the W64R mutation was made in the human beta 3 receptor gene and the resulting mutant receptor expressed in CHO cells. Activation by various agonists showed no significant differences (t-test, P > 0.05) between the wild type and mutant receptors. These studies show that, when expressed in a heterologous system, the W64R mutant receptor is pharmacologically and functionally indistinguishable from the wild type beta 3-adrenergic receptor.

Molecular biology of adrenergic receptors in the rat and frog central nervous system.

Recent developments in the characterization of the adrenergic receptors have led to the identification and purification of the binding subunits of the various catecholamine receptors. beta-Adrenergic receptors have been identified in a wide variety of tissues by photoaffinity labeling with the antagonist [125I]p-azidobenzylcrazolol and have been purified to apparent homogeneity from several of these tissues. Thus, beta 1- and beta 2-adrenergic receptor binding sites appear to reside on peptides with molecular weights of 60,000 to 65,000. The alpha 1-adrenergic receptor binding subunit has been identified in several peripheral tissues by photoaffinity labeling with a newly developed probe (4-amino-6,7-dimethoxy-2[4(5(3-[125I]-iodo-4-azidophenyl) pentanoyl)-1-piperazinyl]-quinazoline, or [125I]APDQ). This binding site resides on a peptide with a molecular weight of 80,000. These techniques have been applied to the elucidation of the binding subunit structure of these receptors in the rat central nervous system with the result that beta 1-, beta 2-, and alpha 1-adrenergic binding sites appear to reside on peptides of similar molecular weight to those identified in peripheral tissues (i.e., 60,000-65,000 and 80,000). Using immunocytochemical techniques with antibodies raised to the frog erythrocyte, beta 2-adrenergic receptor, beta-adrenergic receptors were identified at the light microscopic level in regions of the rat and frog brain previously found by ligand binding and autoradiography to be richest in beta-adrenergic receptors. At the electron microscopic level, beta-receptor immunoreactivity was found throughout dendritic processes with local accumulations at certain postsynaptic sites. This finding is consistent with the idea that the density of the receptors might be significantly increased at postsynaptic junctions of adrenergic neurons.

Specific activation of Gs by synthetic peptides corresponding to an intracellular loop of the beta-adrenergic receptor.

Peptides corresponding to the amino acid sequence of the hamster beta 2-adrenergic receptor (beta 2AR) were synthesized and their ability to activate purified G-proteins determined. Two peptides, comprising the N- and C-terminal 15 amino acids of the putative third intracellular loop region of the beta 2AR were found to activate the G-protein Gs but not to activate a preparation of Gi/Go. Other peptides corresponding to the internal portions of this loop and the C-terminal tail region failed to activate either G-protein. The presence of phospholipid vesicles was required for this activation. The observation that peptides with sequences corresponding to the ends of the third intracellular loop of the beta AR can specifically activate Gs confirms the results of previous mutagenesis studies on the receptor and demonstrates that the secondary structure conferred by the amino acid sequences in these regions is sufficient for the activation of G-proteins.

Genomic organization and functional characterization of the mouse GalR1 galanin receptor.

Galanin mediates diverse physiological functions in digestive, endocrine, and central nervous systems through G-protein-coupled receptors. Two galanin receptors have been cloned but the gene structures are unknown. We report genomic and cDNA cloning of the mouse GalR1 galanin receptor and demonstrate that the coding sequence is uniquely divided into three exons encoding the N-terminal portion through the fifth transmebrane domain, the third intracellular loop, and the sixth transmembrane domain through the C-terminus. Functional analysis of the encoded cDNA revealed active ligand binding and intracellular signaling. The expression is detected in brain, spinal cord, heart and skeletal muscle.

The GalR2 galanin receptor mediates galanin-induced jejunal contraction, but not feeding behavior, in the rat: differentiation of central and peripheral effects of receptor subtype activation.

The neuropeptide galanin mediates a diverse array of physiological functions through activation of specific receptors. Roles of the three recently cloned galanin receptors (GalRs) in rat intestinal contraction and food intake were examined using GalR-selective ligands and the results were compared with the pharmacological profiles of defined GalRs. The action profile of these ligands in jejunal contraction resembled only that of GalR2 and only a high level of GalR2 mRNA was detected in the tissue, supporting GalR2 as the receptor mediating jejunal contraction. The action profile for food intake in rats excluded GalR2, GalR3 and the putative pituitary galanin receptor as the 'feeding receptor', suggesting that either GalR1 or an unidentified GalR is responsible for mediating this function.

Expression of B1 and B2 bradykinin receptor mRNA and their functional roles in sympathetic ganglia and sensory dorsal root ganglia neurones from wild-type and B2 receptor knockout mice.

Bradykinin has been implicated in nociception and inflammation. To examine the relative significance of B1 and B2 bradykinin receptor subtypes in sympathetic and sensory ganglia, the electrophysiological effects of bradykinin analogues and the expression of receptor subtype mRNA were examined in wild-type and "B2 knockout" mice from which the B2 receptor gene had been deleted. In wild-type mice the B2 receptor agonist bradykinin depolarized superior cervical ganglia (SCG) and activated inward currents in dorsal root ganglia (DRG) neurones. Responses to the B1 receptor agonist, [des-Arg10]-kallidin, were seen only in SCG that had been pre-treated with interleukins and the peptidase inhibitor captopril, but not in DRG neurones. The up-regulation of responses to [des-Arg10]-kallidin and substance P were blocked by indomethacin and, thus, were dependent upon cyclo-oxygenase activity. The effects of bradykinin were abolished in SCG and DRG's from B2 knockout mice and this was correlated with the absence of B2 receptor mRNA in ganglia from these animals. However, despite the presence of B1 receptor mRNA in interleukin treated SCG from B2 knockout mice, no depolarizing effects of the B1 receptor agonist [des-Arg10]-kallidin were observed. The successful elimination of bradykinin responses and B2 mRNA in sympathetic and sensory ganglia from B2 knockout mice, confirms that B2 receptors are the predominant functional bradykinin receptor subtype in these tissues and that B1 receptor mRNA is expressed in both sympathetic and sensory ganglia from these animals.

Synthesis of potent cyclic hexapeptide NK-1 antagonists. Use of a minilibrary in transforming a peptidal somatostatin receptor ligand into an NK-1 receptor ligand via a polyvalent peptidomimetic.

The endogenous peptides somatostatin (SRIF) and substance P comprise very different structures. Although both bind G-protein-coupled receptors, the SRIF receptors (SSTR 1-5) recognize SRIF and related peptides which retain its beta-turn such as the potent cyclic hexapeptide SRIF agonist L-363,301 (6a), but not substance P. Conversely the NK-1 receptor binds substance P but not the above ligands. In contrast, the beta-D-glucosides 1 and 2, designed to mimic the beta-turn of 6a, bind both receptors. This observation led us to attempt the conversion of 6a into the first potent, selective cyclic hexapeptide ligand for the NK-1 receptor. To this end, we combined design with a minilibrary approach. The goal was accomplished with surprising ease, leading to the NK-1 receptor antagonist 9 (IC50 2.0 +/- 0.4 nM). This demonstrates that peptidomimetics, incorporating in this case the promiscuous beta-D-glucose scaffold, can provide valuable clues about receptor similarities not revealed by their endogenous ligands. In addition, this work suggests that the use of libraries and rational design need not be mutually exclusive approaches to lead discovery.

Hemocyanin linked to protein A as an immunochemical labeling reagent for electron microscopy.

Hemocyanin-protein A (Hcy/A) is an immunochemical marker suitable for both transmission and scanning electron microscopy. A method is described for the preparation of highly monomeric Hcy/A where protein A and hemocyanin are present in the conjugate in a nearly equal molar ratio. Examples of Hcy/A labeling of several cellular antigens are provided.

Differential effects of intracerebroventricular glucagon-like peptide-1 on feeding and energy expenditure regulation.

Intracerebroventricular (i.c.v.) administration of glucagon-like peptide-1-(7-37) amide (GLP-1) has been shown to modulate food and water intake. The present studies further characterize the effects of i.c.v. GLP-1 in the regulation of energy balance in lean and obese animals. In both obese and lean Zucker rats, a single i.c.v. infusion of GLP-1 (1-30 microg) resulted in a dose-dependent reduction of food intake and decrease in respiratory quotient relative to the saline control during the first 2 h of the nocturnal cycle. In obese Zucker rats, the food intake was reduced by 73 +/- 11% of the control at the 30 microg dose, whereas a modest 45 +/- 18% reduction was observed in lean rats. Despite the large reduction in food intake seen with GLP-1, there was no compensatory decrease in nocturnal oxygen consumption in the obese Zucker rats. Interestingly, low doses of GLP-1 (1 microg) in lean Zucker rats, which had minimal effects on food intake, caused a 19 +/- 7% increase in O2 consumption during the first 2 h of the nocturnal cycle. These data suggest that central GLP-1 may be an important factor controlling negative energy balance in both the lean and obese Zucker rats.

Cytoplasmic loop of beta-adrenergic receptors: synaptic and intracellular localization and relation to catecholaminergic neurons in the nuclei of the solitary tracts.

Pharmacological studies suggest that beta-adrenergic receptors (beta AR) in the medial nuclei of the solitary tracts (m-NTS) facilitate presynaptic release of catecholamines and also function at postsynaptic sites. We have localized the antigenic sites for a monoclonal antibody against a peptide corresponding to amino acids 226-239 of beta AR in the m-NTS of rat brain. By light microscopy, immunoperoxidase labeling for this antibody was detected in somata and proximal processes of many small cells that were distributed throughout the rostrocaudal extent of the m-NTS. Electron microscopy confirmed the cytoplasmic localization of beta AR in perikarya and proximal dendrites of neurons. Immunoreactivity occurred as discrete patches associated with cytoplasmic surfaces of plasma membrane and with irregularly-shaped saccules with clear lumen in the immediate vicinity. Select regions of nuclear envelopes, mitochondrial membranes, and rough endoplasmic reticulum were also immunoreactive along their cytoplasmic surfaces. In contrast, the Golgi apparatus was labeled, but infrequently. Immunoreactivity was also detected at numerous post- and occasional presynaptic membrane specializations of select axodendritic junctions. Dual labeling for the beta AR-antibody by the immunoperoxidase method and for a rabbit antiserum against the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), by the immunoautoradiographic method within the same sections, further established the precise cellular relations between beta AR and catecholaminergic neurons. Immunoreactivity for beta AR was detected in numerous perikarya and proximal dendrites that did not show detectable levels of TH. However, a few cells were dually labeled for both antigens, as seen by both light and electron microscopy. The TH-labeled terminals formed synapses at junctions both with and without beta AR-like immunoreactivity. These results from the single and dual labeling studies: (1) confirm biochemical predictions that amino acids 226-239 of beta AR protein reside intracellularly; (2) provide the first ultrastructural evidence for beta AR localization within both pre- and postsynaptic membrane specializations of a subset of catecholaminergic synapses; and (3) suggest select intracellular sites that may be involved with synthesis and/or internalization and degradation of the receptor protein.

Characterization of the binding and activity of a high affinity, pseudoirreversible morpholino tachykinin NK1 receptor antagonist.

2(S)-((3,5-Bis(trifluoromethyl)benzyl)-oxy)-3(S)-phenyl-4-((3-oxo-1,2,4- triazol-5-yl)methyl)morpholine (L-742,694) is a selective morpholino tachykinin NK1 receptor antagonist that inhibits the binding of 125I-substance P to the human tachykinin NK1 receptor with a Kd = 37 pM. Increasing concentrations of L-742,694 added to cells 15 min prior to agonist progressively increase the apparent EC50 of substance P for inducing the synthesis of inositol phosphate in Chinese hamster ovary (CHO) cells expressing human tachykinin NK1 receptor and decrease the maximal level of stimulation observed. In contrast, addition of substance P and L-742,694 to the cells at the same time results in an increase in the EC50 for substance P with no decrease in the maximal level of stimulation. The compound also decreases the apparent number of binding sites for 125I-substance P observed by Scatchard analysis. Analysis of the binding of [3H]L-742,694 to the tachykinin NK1 receptor shows that it associates with the receptor with k(a) = 3.98 x 10(8) M(-1) min(-1), and dissociates with k(d) = 0.026 min(-1) and t1/2 = 27 min at 22 degrees C. The slow rate of dissociation of L-742,694 from the tachykinin NK1 receptor and the observation that altering the order of addition of antagonist and substance P attenuates the effect of the antagonist on the maximal activation suggest that L-742,694 is a competitive antagonist that can behave as a pseudoirreversible antagonist under some experimental conditions. L-742,694 has reduced affinity for tachykinin NK1 receptors in which alanine has been substituted for Gln165, His197 or His265 in transmembrane helices 4, 5 and 6, respectively. These three residues have previously been shown to be present in the binding site of tachykinin NK1 receptor antagonists of several structural classes. In addition, L-742,694 inhibits binding of the quinuclidine antagonist (2S,3S)-cis-2-(diphenyl methyl)-N-[(2-iodophenyl)-methyl]-1-azabicyclo[2.2.2]octane 3-amine ([125I]L-703,606) with the same affinity as it inhibits binding of 125I-substance P. These data indicate that L-742,694 binds to the same site within the transmembrane domain of the receptor as previously described competitive antagonists.