On the hierarchical classification of G Protein-Coupled Receptors

Motivation: G protein-coupled receptors (GPCRs) play an important role in many physiological systems by transducing an extracellular signal into an intracellular response. Over 50% of all marketed drugs are targeted towards a GPCR. There is considerable interest in developing an algorithm that could effectively predict the function of a GPCR from its primary sequence. Such an algorithm is useful not only in identifying novel GPCR sequences but in characterizing the interrelationships between known GPCRs. Results: An alignment-free approach to GPCR classification has been developed using techniques drawn from data mining and proteochemometrics. A dataset of over 8000 sequences was constructed to train the algorithm. This represents one of the largest GPCR datasets currently available. A predictive algorithm was developed based upon the simplest reasonable numerical representation of the protein's physicochemical properties. A selective top-down approach was developed, which used a hierarchical classifier to assign sequences to subdivisions within the GPCR hierarchy. The predictive performance of the algorithm was assessed against several standard data mining classifiers and further validated against Support Vector Machine-based GPCR prediction servers. The selective top-down approach achieves significantly higher accuracy than standard data mining methods in almost all cases

Identifying Ligand Binding Conformations of the β2-Adrenergic Receptor by Using Its Agonists as Computational Probes

Recently available G-protein coupled receptor (GPCR) structures and biophysical studies suggest that the difference between the effects of various agonists and antagonists cannot be explained by single structures alone, but rather that the conformational ensembles of the proteins need to be considered. Here we use an elastic network model-guided molecular dynamics simulation protocol to generate an ensemble of conformers of a prototypical GPCR, β2-adrenergic receptor (β2AR). The resulting conformers are clustered into groups based on the conformations of the ligand binding site, and distinct conformers from each group are assessed for their binding to known agonists of β2AR. We show that the select ligands bind preferentially to different predicted conformers of β2AR, and identify a role of β2AR extracellular region as an allosteric binding site for larger drugs such as salmeterol. Thus, drugs and ligands can be used as "computational probes" to systematically identify protein conformers with likely biological significance. © 2012 Isin et al

Genetic inhibition of neurotransmission reveals role of glutamatergic input to dopamine neurons in high-effort behavior

Midbrain dopamine neurons are crucial for many behavioral and cognitive functions. As the major excitatory input, glutamatergic afferents are important for control of the activity and plasticity of dopamine neurons. However, the role of glutamatergic input as a whole onto dopamine neurons remains unclear. Here we developed a mouse line in which glutamatergic inputs onto dopamine neurons are specifically impaired, and utilized this genetic model to directly test the role of glutamatergic inputs in dopamine-related functions. We found that while motor coordination and reward learning were largely unchanged, these animals showed prominent deficits in effort-related behavioral tasks. These results provide genetic evidence that glutamatergic transmission onto dopaminergic neurons underlies incentive motivation, a willingness to exert high levels of effort to obtain reinforcers, and have important implications for understanding the normal function of the midbrain dopamine system.Fil: Hutchison, M. A.. National Institutes of Health; Estados UnidosFil: Gu, X.. National Institutes of Health; Estados UnidosFil: Adrover, Martín Federico. National Institutes of Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Lee, M. R.. National Institutes of Health; Estados UnidosFil: Hnasko, T. S.. University of California at San Diego; Estados UnidosFil: Alvarez, V. A.. National Institutes of Health; Estados UnidosFil: Lu, W.. National Institutes of Health; Estados Unido

Expanding the role of tachykinins in the neuroendocrine control of reproduction

Reproductive function is driven by the hormonal interplay between the gonads and brain–pituitary axis. Gonadotropin-releasing hormone (GnRH) is released in a pulsatile manner, which is critical for the attainment and maintenance of fertility; however, GnRH neurons lack the ability to directly respond to most regulatory factors, and a hierarchical upstream neuronal network governs its secretion. We and others proposed a model in which Kiss1 neurons in the arcuate nucleus (ARC), called as KNDy neurons, release kisspeptin (a potent GnRH secretagogue) in a pulsatile manner to drive GnRH pulses under the coordinated autosynaptic action of its cotransmitters, the tachykinin neurokinin B (NKB, stimulatory) and dynorphin (inhibitory). Numerous genetic and pharmacological studies support this model; however, additional regulatory mechanisms (upstream of KNDy neurons) and alternative pathways of GnRH secretion (kisspeptin independent) exist, but remain ill defined. In this aspect, attention to other members of the tachykinin family, namely substance P (SP) and neurokinin A (NKA), has recently been rekindled. Even though there are still major gaps in our knowledge about the functional significance of these systems, substantial evidence, as discussed below, is placing tachykinin signaling as an important pathway for the awakening of the reproductive axis and the onset of puberty to physiological GnRH secretion and maintenance of fertility in adulthood

The differential effects of the gonadotropin receptors on aromatase expression in primary cultures of immature rat granulosa cells are highly dependent on the density of receptors expressed and the activation of the inositol phosphate cascade

Signaling pathways mediating the divergent effects of FSH and LH on aromatase in immature rat granulosa cells were studied by infecting cells with increasing amounts of adenoviral vectors for the hLHR or hFSHR. Increasing amounts of Ad-hLHR, used at a multiplicity of infection (MOI) of 20 or 200 viable viral particles/cell increased hCG binding, hCG-induced cAMP and Akt phosphorylation but inositol phosphates only increased in response to hCG in cells infected with 200 MOI Ad-hLHR. In contrast hCG increased aromatase expression in cells infected with 20 but not in cells infected with 200 MOI Ad-hLHR. Cells infected with 20 or 200 MOI Ad-hFSHR showed increased hFSH binding and hFSH-induced Akt phosphorylation, but the hFSH-induced cAMP response was unchanged relative to control cells. However, hFSH was able to stimulate the inositol phosphate cascade in the Ad-hFSHR infected cells, and the hFSH induction of aromatase was abolished. We also found that activation of C kinase or expression of a constitutively active form of Gαq inhibited the induction of aromatase by hFSH or 8Br-cAMP. We conclude that the differential effects of FSH and LH on aromatase in immature granulosa cells are highly dependent on gonadotropin receptor density and on the signaling pathways activated. We propose that aromatase is induced by common signals generated by activation of the FSHR and LHR (possibly cAMP and Akt) and that the activation of the inositol phosphate cascade in cells expressing a high density of LHR or FSHR antagonizes this induction

Investigating the molecular mechanism of positive and negative allosteric modulators in the calcium-sensing receptor dimer

AbstractAllosteric modulators that are targeting the calcium-sensing receptor (CaSR) hold great therapeutic potential, and elucidating the molecular basis for modulation would thus benefit the development of novel therapeutics. In the present study, we aimed at investigating the mechanism of allosteric modulation in CaSR by testing dimers carrying mutations in the allosteric site of one or both of the subunits. To ensure measurements on a well-defined dimer composition, we applied a trans-activation system in which only the specific heterodimer of two loss-of-function mutants responded to agonist. Although one of these mutants was potentiated by a positive allosteric modulator, we showed that receptor activity was further potentiated in a trans-activation heterodimer containing a single allosteric site, however only when the allosteric site was located in the subunit responsible for G protein coupling. On the contrary, preventing activation in both subunits was necessary for obtaining full inhibition by a negative allosteric modulator. These findings correlate with the proposed activation mechanism of the metabotropic glutamate receptors (mGluRs), in which only a single transmembrane domain is activated at a time. CaSR and mGluRs belong to the class C G protein-coupled receptors, and our findings thus suggest that the activation mechanism is common to this subfamily.</jats:p

Effect of a toggle switch mutation in TM6 of the human adenosine A3 receptor on Gi protein-dependent signalling and Gi-independent receptor internalization

Background and Purpose: The highly conserved tryptophan (W6.48) in transmembrane domain 6 of GPCRs has been shown to play a central role in forming an active conformation in response to agonist binding. We set out to characterize the effect of this mutation on the efficacy of two agonists at multiple signalling pathways downstream of the adenosine A3 receptor. Experimental Approach: Residue W6.48 in the human adenosine A3 receptor fused to yellow fluorescent protein was mutated to phenylalanine and expressed in CHO-K1 cells containing a cAMP response element reporter gene. The effects on agonist-mediated receptor internalization were monitored by automated confocal microscopy and image analysis. Further experiments were carried out to investigate agonist-mediated ERK1/2 phosphorylation, inhibition of [3H]-cAMP accumulation and β-arrestin2 binding. Key Results: NECA was able to stimulate agonist-mediated internalization of the W6.48F mutant receptor, while the agonist HEMADO was inactive. Investigation of other downstream signalling pathways indicated that G-protein coupling was impaired for both agonists tested. Mutation of W6.48F therefore resulted in differential effects on agonist efficacy, and introduced signalling pathway bias for HEMADO at the adenosine A3 receptor. Conclusions and Implications: Investigation of the pharmacology of the W6.48F mutant of the adenosine A3 receptor confirms that this region is important in forming the active conformation of the receptor for stimulating a number of different signalling pathways and that mutations in this residue can lead to changes in agonist efficacy and signalling bias

Design considerations for borehole thermal energy storage (BTES): A review with emphasis on convective heat transfer

Borehole thermal energy storage (BTES) exploits the high volumetric heat capacity of rock-forming minerals and pore water to store large quantities of heat (or cold) on a seasonal basis in the geological environment. The BTES is a volume of rock or sediment accessed via an array of borehole heat exchangers (BHE). Even well-designed BTES arrays will lose a significant quantity of heat to the adjacent and subjacent rocks/sediments and to the surface; both theoretical calculations and empirical observations suggest that seasonal thermal recovery factors in excess of 50% are difficult to obtain. Storage efficiency may be dramatically reduced in cases where (i) natural groundwater advection through the BTES removes stored heat, (ii) extensive free convection cells (thermosiphons) are allowed to form, and (iii) poor BTES design results in a high surface area/volume ratio of the array shape, allowing high conductive heat losses. The most efficient array shape will typically be a cylinder with similar dimensions of diameter and depth, preferably with an insulated top surface. Despite the potential for moderate thermal recovery, the sheer volume of thermal storage that the natural geological environment offers can still make BTES a very attractive strategy for seasonal thermal energy storage within a “smart” district heat network, especially when coupled with more efficient surficial engineered dynamic thermal energy stores (DTES)