Chemical Information Transfer Research Articles

(Invited) Nanosensor Coupling to Human and Plant Interfaces for Real Time Chemical Information Transfer

Our laboratory at MIT has been interested over the past few years in new techniques to facilitate the transfer of chemical information from living organisms, specifically plants, animals and humans, for applications ranging from precision agriculture to precision medicine. This presentation will discuss recent advances on this topic. As tool towards this end, fluorescent nanosensors hold the potential to revolutionize life sciences and medicine. However, their adaptation and translation into the in vivo environment is fundamentally hampered by unfavourable tissue scattering and intrinsic autofluorescence. Here we develop wavelength-induced frequency filtering (WIFF) whereby the fluorescence excitation wavelength is modulated across the absorption peak of a nanosensor, allowing the emission signal to be separated from the autofluorescence background, increasing the desired signal relative to noise, and internally referencing it to protect against artefacts. Using highly scattering phantom tissues, an SKH1-E mouse model and other complex tissue types, we show that WIFF improves the nanosensor signal-to-noise ratio across the visible and near-infrared spectra up to 52-fold. This improvement enables the ability to track fluorescent carbon nanotube sensor responses to riboflavin, ascorbic acid, hydrogen peroxide and a chemotherapeutic drug metabolite for depths up to 5.5 ± 0.1 cm when excited at 730 nm and emitting between 1,100 and 1,300 nm, even allowing the monitoring of riboflavin diffusion in thick tissue. As an application, nanosensors aided by WIFF detect the chemotherapeutic activity of temozolomide transcranially at 2.4 ± 0.1 cm through the porcine brain without the use of fibre optic or cranial window insertion. The ability of nanosensors to monitor previously inaccessible in vivo environments will be important for life-sciences research, therapeutics and medical diagnostics. Also towards this overall objective, our laboratory at MIT has been interested in exploring the relatively new interface between living plants and non-biological nanostructures to impart the former with new and enhanced functions, which we call Plant Nanobionics. We have developed a theory of subcellular uptake and kinetic trapping of a wide range of nanoparticles, validated in-vivo in living plants. Confocal visible and near infrared fluorescent microscopy and single particle tracking of Gold-Cystein-AF405 (GNP-Cys-AF405), Streptavidin-Quantum Dot (SA-QD), Dextran and Poly(acrylic acid) nanoceria, and various polymer-wrapped SWCNT, including lipid-PEG-SWCNT, chitosan-SWCNT and (AT)15-SWCNT, were used to demonstrate that particle size and the magnitude, but not the sign, of the zeta potential are key in determining whether a particle is spontaneously and kinetically trapped within chloroplasts or the cytosol. We develop a mathematical model of this Lipid Exchange Envelope Penetration (LEEP) mechanism, which agrees well with observations of this size and zeta potential dependence. As an application, we rationally designed a chitosan-complexed single-walled carbon nanotube (SWNT) as nanocarriers to selectively deliver plasmid DNA (pDNA) to chloroplasts of different plant species without external biolistic or chemical aid. We demonstrate chloroplast-targeted transgene delivery and expression in living mature arugula (Eruca sativa) and watercress (Nasturitium officinale) plants in planta and in isolated Arabidopsis thaliana mesophyll protoplasts. Another application of nanoparticles and nanotechnology to plant sciences is in the form of biochemical sensors that operate in planta and across diverse species. Using non-destructive optical nanosensors, we find that the spatial and temporal H2O2 concentration immediately post-wounding follows a simple logistic waveform for six dicot plant species: lettuce (Lactuca sativa), arugula (Eruca sativa), spinach (Spinacia oleracea), strawberry blite (Blitum capitatum), sorrel (Rumex acetosa), and Arabidopsis thaliana, ranked in order of wave speed from 0.44 to 3.10 cm/min. The H2O2 wave tracks the concomitant surface potential wave measured electrochemically for the series of plants. We show that the plant NADPH oxidase RbohD, glutamate receptor-like channels (GLR3.3 and GLR3.6) are all critical to the propagation of the H2O2 waveform upon wounding. Our findings highlight the utility of a new type of nanosensor probe that is species-independent and capable of real-time, spatial and temporal biochemical measurements in planta.

Integration of multiple volatile cues into plant defense responses.

The ability to predict future risks is essential for many organisms, including plants. Plants can gather information about potential future herbivory by detecting volatiles that are emitted by herbivore-attacked neighbors. Several individual volatiles have been identified as active danger cues. Recent work has also shown that plants may integrate multiple volatiles into their defense responses. Here, I discuss how the integration of multiple volatiles can increase the capacity of plants to predict future herbivore attack. I propose that integration of multiple volatile cues does not occur at the perception stage, but may through downstream early defense signaling and then be further consolidated by hormonal crosstalk. Exploring plant volatile cue integration can facilitate our understanding and utilization of chemical information transfer.

Functional Characterization of Chemosensory Protein AmalCSP5 From Apple Buprestid Beetle, Agrilus mali (Coleoptera: Buprestidae).

In the sensitive and complex chemo-sensation system of insects, chemosensory proteins (CSPs) can facilitate the transfer of chemical information and play important roles for variable behaviors of insects. We cloned the chemosensory protein AmalCSP5 from antennae of the apple buprestid beetle (Agrilus mali Matsumura), a serious invasive pest of wild apple trees. Expression profiling showed that AmalCSP5 was expressed in various tissues, suggesting its significance in multiple physiological activities and behaviors of A. mali. AmalCSP5 was preferentially expressed in female antennae and male abdomens. AmalCSP5 was able to bind a variety of test volatiles, especially alcohols and esters. AmalCSP5 exhibited good binding affinity for all five test secondary compounds (i.e., procyanidin, phlorizin, kaemferol, chlorogenic acid, and rutin), suggesting its preferential binding abilities to nonvolatile host plant secondary metabolites and critical roles in gustatory perception of nonvolatiles. Tyr27 and Ser69 of AmalCSP5 could form hydrogen bonds with hexyl benzoate and hexyl hexanoate, respectively. Procyanidin, the best ligand among all test compounds, could form hydrogen bonds with three amino acid residues (i.e., Arg7, Leu8, and Lys41) of AmalCSP5. Thus, high ligand binding affinity for AmalCSP5 seemed to be dependent mainly on the formation of hydrogen bonds. The putative key amino acid residues of AmalCSP5 can be used as molecular targets for designing and screening new attractants and repellents for A. mali. Our results provide insights into binding interactions of AmalCSP5 with volatile and nonvolatile ligands, and a firm basis for developing eco-friendly management strategies of A. mali.

Plant Secondary Metabolite Diversity and Species Interactions

Ever since the first plant secondary metabolites (PSMs) were isolated and identified, questions about their ecological functions and diversity have been raised. Recent advances in analytical chemistry and complex data computation, as well as progress in chemical ecology from mechanistic to functional and evolutionary questions, open a new box of hypotheses. Addressing these hypotheses includes the measurement of complex traits, such as chemodiversity, in a context-dependent manner and allows for a deeper understanding of the multifunctionality and functional redundancy of PSMs. Here we review a hypothesis framework that addresses PSM diversity on multiple ecological levels (α, β, and γ chemodiversity), its variation in space and time, and the potential agents of natural selection. We use the concept of chemical information transfer as mediator of antagonistic and mutualistic interaction to interpret functional and microevolutionary studies and create a hypothesis framework for understanding chemodiversity as a factor driving ecological processes.

Chemical Communication between Molecules.

In this Minireview conceptual approaches towards the communication between (supra)molecular species are illustrated. The reviewed mechanisms include photophysical communication through energy transfer and communication mediated by chemical species that are released from precursors upon application of an external stimulus. The focus is on the stimulation by (reversible) photoreactions. Special attention has been given to supramolecular systems that illustrate the idea of chemical communication and information transfer in a very descriptive manner.

Changing odour landscapes: the effect of anthropogenic volatile pollutants on plant–pollinator olfactory communication

Summary Olfactory signals, often in synergy with visual signals, mediate the interactions between plants and animals. However, urbanization and agricultural practices are both sources of volatile organic compounds (VOCs) and reactive oxygen species (ROS) that have the potential to interfere with plant–animal communication and to disrupt mutualistic interactions. In this review, we explore how anthropogenic airborne pollutants may disrupt chemical information transfer between flowering plants and flower visitors. The emission of anthropogenic volatile pollutants (AVPs) including VOCs and formation of ROS, for example from traffic or industries, and non‐natural biogenic VOCs, for example from introduced crops, may have a number of effects: (i) changes in plant signalling as a consequence of plants experiencing physiological stress; (ii) chemical interference (chemical degradation/transformation of infochemicals); (iii) increased levels of background noise impeding signal detection; and (iv) changes in pollinator signal perception and behaviour. All of the above in turn could have consequences for the biological fitness of plants and animals that rely on olfactory information as pivotal functional signals. The study of anthropogenic airborne pollutants and their effects on plant signalling is just emerging and the impacts of this aspect of anthropogenic emissions are barely understood. Volatiles emitted from anthropogenically changed landscapes could, however, have far‐reaching consequences for ecosystem functioning in adjacent natural zones, particularly in fragmented landscapes. In response to the wide gap in our knowledge on the mechanisms that govern interference of anthropogenic VOCs with olfactory information, future research directions are proposed with the aim to inspire research to help elucidate the risks of anthropogenic VOCs for plant–pollinator communities and improve risk assessment strategies. A lay summary is available for this article.

Cuticular Hydrocarbons as Potential Close Range Recognition Cues in Orchid Bees.

Male Neotropical orchid bees collect volatile chemicals from their environment and compose species-specific volatile signals, which are subsequently exposed during courtship display. These perfumes are hypothesized to serve as attractants and may play a role in female mate choice. Here, we investigated the potential of cuticular hydrocarbons as additional recognition cues. The cuticular hydrocarbons of males of 35 species belonging to four of the five extant euglossine bee genera consisted of aliphatic hydrocarbons ranging in chain lengths between 21 and 37 C-atoms in distinct compositions, especially between sympatric species of similar coloring and size, for all but one case. Cleptoparasitic Exaerete spp. had divergent profiles, with major compounds predominantly constituted by longer hydrocarbon chains (>30 C-atoms), which may represent an adaptation to the parasitic life history ("chemical insignificance"). Phylogenetic comparative analyses imply that the chemical profiles exhibited by Exaerete spp. are evolutionarily divergent from the rest of the group. Female hydrocarbon profiles were not identical to male profiles in the investigated species, with either partial or complete separation between sexes in multivariate analyses. Sexually dimorphic hydrocarbon profiles are assumed to be the basis for sex recognition in a number of insects, and thus may supplement the acquired perfume phenotypes in chemical information transfer. Overall, cuticular hydrocarbons meet the requirements to function as intraspecific and intersexual close range recognition signals; behavioral experiments are needed to determine their potential involvement in mate recognition.

New Lives for Seven Transmembrane Receptors as Drug Targets

Historically, seven transmembrane receptors (7TMs) [also referred to as G-protein-coupled receptors (GPCRs)] have been the mainstay biological target for pharmacological therapy. This is because they are conveniently located on the cell surface, and thus are the main points of transfer of chemical information from the extracellular space to the cytosol, and they control a myriad of cellular processes. This latter point becomes a two-edged sword in the utilization of 7TMRs as drug targets since it then requires some form of limitation of receptor signaling to achieve therapeutic drug selectivity.

The scent of wolves: pyrazine analogs induce avoidance and vigilance behaviors in prey.

The common gray wolf (Canis lupus) is an apex predator located at the top of the food chain in the Northern Hemisphere. It preys on rodents, rabbits, ungulates, and many other kinds of mammal. However, the behavioral evidence for, and the chemical basis of, the fear-inducing impact of wolf urine on prey are unclear. Recently, the pyrazine analogs 2, 6-dimethylpyrazine, 2, 3, 5-trimethylpyrazine and 3-ethyl-2, 5-dimethyl pyrazine were identified as kairomones in the urine of wolves. When mice were confronted with a mixture of purified pyrazine analogs, vigilance behaviors, including freezing and excitation of neurons at the accessory olfactory bulb, were markedly increased. Additionally, the odor of the pyrazine cocktail effectively suppressed the approach of deer to a feeding area, and for those close to the feeding area elicited fear-related behaviors such as the “tail-flag,” “flight,” and “jump” actions. In this review, we discuss the transfer of chemical information from wolf to prey through the novel kairomones identified in wolf urine and also compare the characteristics of wolf kairomones with other predator-produced kairomones that affect rodents.

Gastropod grazing on a benthic alga leads to liberation of food‐finding infochemicals

Chemical information transfer is a major agent in the regulation of interspecific and intraspecific interactions in natural ecosystems. One important group of such infochemicals both in terrestrial and aquatic ecosystems are so‐called volatile organic compounds (VOCs) that can evoke behavioral or physiological responses like predator avoidance and mate or host location. In previous work, we have demonstrated that freshwater gastropods utilize VOCs released from benthic algae as food finding cues, although the specific nature of the VOC release and perception were not yet clear. Therefore we tested whether gastropod grazing on biofilms leads to algal cell damage and a subsequent liberation of wounding‐associated VOCs. In bioassays we investigated the algal VOC bouquet level which is necessary to elicit a behavioural response of freshwater gastropods. The results of the liberation experiment showed that gastropod grazing leads to VOCs release. We also found that a certain threshold level of volatiles is necessary for snails to recognise the volatile infochemicals and subsequently respond with a directed foraging behaviour towards the odour. Finally, a calculated mass balance model demonstrated that the grazer mediated VOC release produced a signal concentration that is sufficient to be recognized by conspecifics and utilized as foraging infochemicals. The emission of ecologically relevant volatiles through snail grazing with subsequent attraction of other gastropod grazers to algal biofilms indicates an important but so far understudied chemical signaling mechanism of ecological importance.

Enzymatic Writing to Soft Films: Potential to Filter, Store, and Analyze Biologically Relevant Chemical Information

Sensor‐based chemical analyses commonly enlist either the molecular recognition capabilities of biology (e.g., enzyme biosensors) or advanced information processing algorithms (e.g., the electronic nose). Here, a hybrid approach is proposed in which an enzyme is used to “filter” chemical information and write this information to a film which then serves as a permanent storage medium that can be ‘read’ repeatedly, interactively, and by multiple sensor modalities. This approach is demonstrated by analyzing common dietary phenols that are reported to offer health benefits. Specifically, the enzyme tyrosinase is used to convert these phenols into reactive quinones that graft (i.e., write) to a chitosan film. Grafting can be detected by optical, mechanical, and electrochemical sensors. Importantly, grafting confers redox activity to the films and this redox activity can be probed interactively by advanced electrochemical methods that allow the intrinsic redox reactivities to be compared, redox interactions to be identified, and biologically relevant redox activities to be examined. The transfer of chemical and biological information to a film is envisioned to provide broader access to the extensive capabilities offered by sensor technologies and signal processing methodologies.

Ladderphanes: A New Type of Duplex Polymers

A polymeric ladderphane is a step-like structure comprising multiple layers of linkers covalently connected to two or more polymeric backbones. The linkers can be planar aromatic, macrocyclic metal complexes, or three-dimensional organic or organometallic moieties. Structurally, a DNA molecule is a special kind of ladderphane, where the cofacially aligned base-pair pendants are linked through hydrogen bonding. A greater understanding of this class of molecules could help researchers develop new synthetic molecules capable of a similar transfer of chemical information. In this Account, we summarize our studies of the strategy, design, synthesis, characterization, replications, chemical and photophysical properties, and assembly of a range of double-stranded ladderphanes with many fascinating structures. We employed two norbornene moieties fused with N-arylpyrrolidine to connect covalently with a range of relatively rigid linkers. Ring opening metathesis polymerizations (ROMP) of these bis-norbornenes using the first-generation Grubbs ruthenium-benzylidene catalyst produced the corresponding symmetrical double-stranded ladderphanes. The N-arylpyrrolidene moiety in the linker controls the isotactic selectivity and the trans configuration for all double bonds in both single- and double-stranded polynorbornenes. The π-π interactions between these aryl pendants may contribute to the high stereoselectivity in the ROMP of these substrates. We synthesized chiral helical ladderphanes by incorporating asymmetric center(s) in the linkers. Replication protocols and sequential polymerization of a monomer that includes two different polymerizable groups offer methods for producing unsymmetical ladderphanes. These routes furnish template synthesis of daughter polymers with well-controlled chain lengths and polydispersities. The linkers in these ladderphanes are well aligned in the center along the longitudinal axis of the polymer. Fluorescence quenching, excimer formation, or Soret band splitting experiments suggest that strong interactions take place between the linkers. The antiferromagnetism of the oxidized ferrocene-based ladderphanes further indicates strong coupling between linkers in these ladderphanes. These polynorbornene-based ladderphanes can easily aggregate to form a two-dimensional, highly ordered array on the graphite surface with areas that can reach the submicrometer range. These morphological patterns result from interactions between vinyl and styryl end groups via π-π stacking along the longitudinal axis of the polymer and van der Waals interaction between backbones of polymers. Such assembly orients planar arene moieties cofacially, and polynorbornene backbones insulate each linear array of arenes from the adjacent arrays. Dihydroxylation converts the double bonds in polynorbornene backbones of ladderphanes into more hydrophilic polyols. Hydrogen bonding between these polyol molecules leads to self-assembly and produces structures with longitudinally staggered morphologies on the graphite surface.

The origin and dynamic evolution of chemical information transfer

Although chemical communication is the most widespread form of communication, its evolution and diversity are not well understood. By integrating studies of a wide range of terrestrial plants and animals, we show that many chemicals are emitted, which can unintentionally provide information (cues) and, therefore, act as direct precursors for the evolution of intentional communication (signals). Depending on the content, design and the original function of the cue, there are predictable ways that selection can enhance the communicative function of chemicals. We review recent progress on how efficacy-based selection by receivers leads to distinct evolutionary trajectories of chemical communication. Because the original function of a cue may channel but also constrain the evolution of functional communication, we show that a broad perspective on multiple selective pressures acting upon chemicals provides important insights into the origin and dynamic evolution of chemical information transfer. Finally, we argue that integrating chemical ecology into communication theory may significantly enhance our understanding of the evolution, the design and the content of signals in general.

Molecular motors as an auto‐oscillator

The organization of biomotile systems possesses structural and functional hierarchy, building up from single molecules via protein assemblies and cells further up to an organ. A typical example is the hierarchy of cardiac muscle, on the top of which is the heart. The heartbeat is supported by the rhythmic contraction of the muscle cells that is controlled by the Ca(2+) oscillation triggered by periodic electrical excitation of pacemaker cells. Thus, it is usually believed that the heartbeat is governed by the control system based on a sequential one-way chain with the electrical∕chemical information transfer from the upper to the lower level of hierarchy. On the other hand, it has been known for many years that the contractile system of muscle, i.e., skinned muscle fibers and myofibrils, itself possesses the auto-oscillatory properties even in the constant chemical environment. A recent paper [Plaçais, et al. (2009), Phys. Rev. Lett. 103, 158102] demonstrated the auto-oscillatory movement∕tension development in an in vitro motility assay composed of a single actin filament and randomly distributed myosin II molecules, suggesting that the auto-oscillatory properties are inherent to the contractile proteins. Here we discuss how the molecular motors may acquire the higher-ordered auto-oscillatory properties while stepping up the staircase of hierarchy.

Third International Colloquium on Physical and Chemical Information Transfer in Regulation of Reproduction and Ageing: September 22-28 · 1980 Varna, Bulgaria

AndrologiaVolume 12, Issue 4 p. 337-337 Third International Colloquium on Physical and Chemical Information Transfer in Regulation of Reproduction and Ageing: September 22–28 · 1980 Varna, Bulgaria First published: July‐August 1980 https://doi.org/10.1111/j.1439-0272.1980.tb00643.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume12, Issue4July‐August 1980Pages 337-337 RelatedInformation

Third International Colloquium on Physical and Chemical Information Transfer in Regulation of Reproduction and Ageing: September 22-28 · 1980 Varna, Bulgaria

Third International Colloquium on Physical and Chemical Information Transfer in Regulation of Reproduction and Ageing: September 22-28 · 1980 Varna, Bulgaria

Ligand-Receptor Communication and Drug Design

Ligand-protein and protein-protein interactions play a pivotal role in any cellular process and function by means of complex and dynamic mechanisms that involve sophisticated intra- and intermolecular communication pathways. The deeper understanding of the molecular and structural mechanisms of these pathways of chemical information transfer constitutes the foundations of rational druggable target discovery and drug design. In this context the role of both molecular recognition/communication between the interacting partners and their quantitative/dynamic description constitute the crucial point. In this respect, many approaches at different level of complexity have been developed and applied to different druggable target like enzymes, membrane receptors and protein assembly. They mainly differ in the accuracy and resolution level of molecular description and, hence, in the derived quantitative molecular descriptors/predictors and ligand-target models. In this review, we will try to illustrate some selected examples of ligand-target receptor protein models, by comparatively considering both series of ligands (ligand-based communication modeling) and ligand-target complexes (target-based communication modeling) in order to describe the relevant structural/dynamic features of chemical information transfer in the ligand/drug design endeavour.

Induced response against herbivory by chemical information transfer between plants

Plants respond to herbivores and pathogens attack with increased emission of volatile organic compounds. These molecules act as indirect defences when attracting natural enemies of herbivores and thus benefit the plant. It remains controversial whether undamaged plants capture chemicals released by damaged neighbouring plants and respond to them by increasing their defensive barriers against an imminent attack. In spite of public appeal and of this being the 25th year of the Talking Trees Hypothesis, only recently have the most sceptical scientists been convinced. The induced response to herbivory by interplant information transfer has been found in two plant-herbivore systems. However, the universality of the phenomenon and its ecological and evolutionary relevance remain unclear. The integration of Molecular Biology, Biochemistry, Physiology, and Ecology begin to shed light on the mechanisms of the signal transfer. This integrative approach has developed new and more sensitive tools for identification of complex volatile mixtures and for understanding the process of biosynthesis, emission, transportation and reception of signals.

Enteric neuroimmunophysiology and pathophysiology1,2

Minute-to-minute behavior of the bowel, whether it is normal or disordered, is determined by integrative functions of the enteric nervous system (ENS). Information input processed by the ENS is derived from local sensory receptors, the central nervous system, and immune/inflammatory cells including mast cells. Enteric mast cells use the power of the immune system for detection of antigenic threats and for long-term memory of the identity of the specific antigens. Specific antibodies attach to the mast cells and enable the mast cell to detect sensitizing antigens when they reappear in the gut lumen. Should the sensitizing antigen reappear, mast cells detect it and signal its presence to the ENS. The ENS interprets the mast cell signal as a threat and calls up from its program library secretory and propulsive motor behavior that is organized to eliminate the threat rapidly and effectively. Operation of the alarm program protects the individual, but at the expense of symptoms that include cramping abdominal pain, fecal urgency, and diarrhea. Enteric mast cells use immunologic memory functions to detect foreign antigens as they appear and reappear throughout the life of the individual. Mast cells use paracrine signaling for the transfer of chemical information to the neural networks of the ENS. Integrative circuits in the ENS receive and interpret the chemical signals from the mast cells. Signals from the mast cells are interpreted by the ENS as a labeled code for the presence of a threat in the intestinal lumen.

Structural features of the inactive and active states of the melanin-concentrating hormone receptors: insights from molecular simulations.

Comparative molecular dynamics simulations of both subtypes 1 and 2 of the melanin-concentrating hormone receptor (MCHR1 and MCHR2, respectively) in their free and hormone-bound forms have been carried out. The hormone has been used in its full-length and truncated forms, as well as in 16 mutated forms. Moreover, MCHR1 has been simulated in complex with T-226296, a novel orally active and selective antagonist. The comparative analysis of an extended number of receptor configurations suggests that the differences between inactive (i.e., free and antagonist-bound) and active (i.e., agonist-bound) states of MCHRs involve the receptor portions close to the E/DRY and NPxxY motifs, with prominence to the cytosolic extensions of helices 2, 3, 6, and 7. In fact, the active forms of these receptors share the release of selected intramolecular interactions found in the inactive forms, such as that between R3.50 of the E/DRY motif and D2.40, and that between Y7.53 of the NPxxY motif and F7.60. Another feature of the active forms of both MCHRs is the approach of "helix 8" to the cytosolic extension of helix 3. These features of the active forms are concurrent with the opening of a cleft at the cytosolic end of the helix bundle. For both MCHRs, the agonist-induced chemical information transfer from the extracellular to the cytosolic domains is mediated by a cluster of aromatic amino acids in helix 6, following the ligand interaction with selected amino acids in the extracellular half of the receptor.