Specific receptors for lutropin (luteinizing hormone; LH) and follitropin (follicle-stimulating hormone; FSH) mediate the actions of human chorionic gonadotropin (hCG) and FSH5 on the gonads. Here we report that short independent sequences of the beta-subunit enable hCG to distinguish between the receptors for FSH and LH. Residues between the 11th and 12th cysteines restrict FSH receptor binding; residues between the 10th and 11th cysteines and, to a much lesser extent, residues carboxy-terminal to the 12th cysteine also affect LH receptor binding. CF101-109, an hCG analogue containing hFSH beta residues between the 11th and 12th cysteines, had high affinity for both LH and FSH receptors. Modifications to CF101-109 that reduce binding to either LH or FSH receptors yield gonadotropin analogues having differing ratios of LH:FSH activity. Ligand-binding specificity of the LH receptor is determined by residues encoded by parts of exons 2-4 and 7-9 which prevent hFSH binding but have little effect on hCG binding. FSH receptor specificity is controlled primarily by residues encoded by exons 5 and 6 that prevent hCG binding but have little effect on hFSH binding. These determinants can be interchanged to create receptor analogues that bind hCG and hFSH. Our observations support a model in which distinct negative determinants restrict ligand-receptor interaction. This explains coevolution of binding specificity in families of homologous ligands and their receptors. Natural or designed manipulation of these determinants leads to the 'evolution' of new, specific protein-protein interactions.

The goal of these studies was to devise a model that explains how human chorionic gonadotropin (hCG) interacts with lutropin (LH) receptors to elicit a hormone signal. Here we show that alpha-subunit residues near the N terminus, the exposed surface of the cysteine knot, and portions of the first and third loops most distant from the beta-subunit interface were recognized by antibodies that bound to hCG-receptor complexes. These observations were combined with similar data obtained for the beta-subunit (Cosowsky, L., Rao, S.N.V., Macdonald, G.J., Papkoff, H., Campbell, R.K., and Moyle, W.R. (1995) J. Biol. Chem. 270, 20011-20019), information on residues of hCG that can be changed without disrupting hormone function, the crystal structure of deglycosylated hCG, and the crystal structure of a leucine-repeat protein to devise a model of hCG-receptor interaction. This model suggest that the extracellular domain of the LH receptor is "U-" or "J"-shaped and makes several contacts with the transmembrane domain. High affinity hormone binding results from interactions between residues in the curved portion of the extracellular domain of the receptor and the groove in the hormone formed by the apposition of the second alpha-subunit loop and the first and third beta-subunit loops. Most of the remainder of the hormone is found in the large space between the arms of the extracellular domain and makes few, if any, additional specific contacts with the receptor needed for high affinity binding. Signal transduction is caused by steric or other influences of the hormone on the distance between the arms of the extracellular domain, an effect augmented by the oligosaccharides. Because the extracellular domain is coupled at multiple sites to the transmembrane domain, the change in conformation of the extracellular domain is relayed to the transmembrane domain and subsequently to the cytoplasmic surface of the plasma membrane. While the model does not require the hormone to contact the transmembrane domain to initiate signal transduction, small portions of both subunits may be near the transmembrane domain and assist in initiating the hormonal signal. This is the first model that is consistent with all known information on the activity of the gonadotropins including the amounts of the hormone that are exposed in the hormone-receptor complex, the apparent lack of specific contacts between much of the hormone and the receptor, and the roles of the oligosaccharides in signal transduction.(ABSTRACT TRUNCATED AT 400 WORDS)

Retinal bipolar cells are known to form a complex, interconnecting network through electrical synapses that are either heterologous (with amacrine cells) or homologous (with other bipolar cells). These electrical synapses can be functionally as important as chemical synapses because their distinct properties provide a different character for the network. Much less is known, however, about electrical synapses in retinal bipolar cells than about chemical synapses. Here bipolar cell ͉ cx36 ͉ cx45 ͉ gap junction ͉ AII amacrine cell B ipolar cells, the interneurons that relay information from photoreceptors to ganglion cells in the retina, are essential for visual information processing. The flow of visual information over separate parallel pathways, such as the ON͞OFF and transient͞ sustained pathways, depends on various subtypes of bipolar cells that employ both chemical and electrical synapses to communicate with other neurons in the retina. In the past, most research on the role of bipolar cells focused on their chemical synapses, but it has been known for more than two decades that electrical synapses are also used for signaling by these cells (1-6). In fact, these electrical synapses seem to be an essential component of certain retinal circuits, such as the rod pathway (7). To fully understand information processing in retinal bipolar cells, then, the properties of electrical synapses must be elucidated.Electrical synapses, also called ''gap junctions,'' are formed by proteins called connexins that belong to a family composed of Ͼ20 members (20 connexins in mouse and 21 in human have been cloned so far). Different connexins have been shown to endow gap junctions with different properties, such as conductance, pH modulation, calcium modulation, etc. Because these functional properties depend on the molecular components that make up an electrical synapse, it is important to know which connexins are used by which retinal neurons. Thus, discovering the possible candidate connexins expressed in the bipolar cells has recently been an active area of research.Precisely localizing connexins to bipolar cells, however, turns out to be very difficult. First, the immunolabeled connexins appear as puncta in the inner plexiform layer (IPL), and it is impossible to identify retinal neurons from just these puncta. Second, there are at least 10 types of bipolar cells in the mouse retina (8, 9), and the presence of so many subtypes makes sorting out the expression pattern of connexins even more difficult. Third, connexins are located at the junctions between two adjacent cells, and the resolution of light microscopy (even confocal microscopy) is close to the limit for determining which cellular partner contributes connexin immunostaining at those junctions.Here we examine the expression pattern of connexins in ON cone bipolar cells. Our goal is to overcome the three problems noted above by using single-cell RT-PCR and immunocytochemistry and also by making use of a line of transgenic mice (GUS-GFP) in which a specific type of O...

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Physiological and pharmacological properties of possible subtypes of the native glycine receptor were investigated in retinal neurons using whole-cell voltage-clamp techniques. Two discrete inhibitory glycine responses were identified in ganglion cells. The responses could be distinguished pharmacologically: one was sensitive to strychnine and the other to 5,7-dichlorokynurenic acid. The two responses had different kinetics: the former had a fast onset and fast desensitization, whereas the latter had a slower onset and was much more sustained. The physiological and pharmacological distinctions suggest that the responses are mediated by different receptors. These receptors transduce glycinergic synaptic signals to ganglion cells, where they serve as low-and high-pass filters, respectively, of EPSPs.Key words : glycine receptors; inhibition; strychnine; 5,7-dichlorokynurenic acid; ganglion cells; retina In the vertebrate retina, glycine and GABA share the task of mediating inhibition to ganglion cells. They contribute to the formation of trigger features, such as directional selectivity and edge detection (Caldwell et al., 1978). Several GABA receptor subtypes have been identified and linked to specific aspects of visual information processing Pan and Slaughter, 1991;Zhang and Slaughter, 1995). Although GABAergic and glycinergic neurons are equally populous in the inner retina, a similar diversity of glycinergic receptors has not been described.There is reason to suspect that discrete glycine receptor subtypes exist in retina. Like the GABA receptor, the glycine receptor is a pentamer of ␣ and ␤ subunits in which there are multiple isoforms. In mammalian retina, three ␣ subunit isoforms (␣ 1 , ␣ 2 , ␣ 3 ) have been localized to rat ganglion cells (Greferath et al., 1994). This molecular diversity implies functional and pharmacological variability (Becker, et al., 1988;Betz, 1991;Malosio, 1991a); however, it has proven difficult to translate molecular studies to properties of native glycine receptors or to determine the physiological significance of differential expression.We examined native glycine receptors in isolated amphibian retinal neurons and found that glycine produced two currents: a large, fast, transient, desensitizing component and a smaller, slower, sustained component. Selective antagonists of each of these two currents were identified, implicating two subtypes of the glycine receptor. The agonist and antagonist sensitivities of these two putative receptors were characterized, and their role in synaptic transmission was identified. The results indicate that tonic and phasic glycinergic IPSPs result from two populations of receptor. MATERIALS AND METHODSAnimal experimental preparation. The isolated retinal cell preparation has been described (Bader et al., 1979;Pan and Slaughter, 1995). Briefly, the tiger salamander Ambystoma tigrinum (Kons Scientific, Germantown, WI) was decapitated and pithed, and the eyes were removed. The retina was isolated and incubated for ϳ30 -60 min at room temperature (22...

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Trustworthy operation of industrial control systems depends on secure and real-time code execution on the embedded programmable logic controllers (PLCs). The controllers monitor and control the critical infrastructures, such as electric power grids and healthcare platforms, and continuously report back the system status to human operators. We present Zeus, a contactless embedded controller security monitor to ensure its execution control flow integrity. Zeus leverages the electromagnetic emission by the PLC circuitry during the execution of the controller programs. Zeus's contactless execution tracking enables non-intrusive monitoring of security-critical controllers with tight real-time constraints. Those devices often cannot tolerate the cost and performance overhead that comes with additional traditional hardware or software monitoring modules. Furthermore, Zeus provides an air-gap between the monitor (trusted computing base) and the target (potentially compromised) PLC. This eliminates the possibility of the monitor infection by the same attack vectors.Zeus monitors for control flow integrity of the PLC program execution. Zeus monitors the communications between the humanmachine interface and the PLC, and captures the control logic binary uploads to the PLC. Zeus exercises its feasible execution paths, and fingerprints their emissions using an external electromagnetic sensor. Zeus trains a neural network for legitimate PLC executions, and uses it at runtime to identify the control flow based on PLC's electromagnetic emissions. We implemented Zeus on a commercial Allen Bradley PLC, which is widely used in industry, and evaluated it on real-world control program executions. Zeus was able to distinguish between different legitimate and malicious executions with 98.9% accuracy and with zero overhead on PLC execution by design.