Functional characterisation of A2A receptor thermostable mutants using a yeast signalling assay

G-protein-coupled receptors (GPCR) are transmembrane proteins that play a crucial role in the communication of cells with their external environment. In the last few years, several GPCR crystal structures have been solved using genetically engineered protein. The turkey β1-adrenergic receptor, the human neutrotensin 1 receptor and the adenosine A2A receptor (A2AR) structures involved the introduction of stabilizing mutations. The engineered mutant can be stabilized in an agonist or an antagonist bound conformation making the GPCR less flexible and therefore easier to crystallize. The aim of this study was to use functional characterization of the key thermostabilising mutants of the A2AR in order to understand the molecular basis of the thermostabilisation. The different mutants were characterized using a yeast-based growth assay, which measures down-stream signaling in response to agonist and radioligand binding analysis using both an agonist and an antagonist. Point mutations leading to a reduction/loss of constitutive receptor activity have been identified. In addition, a single point mutation abolishing the ability of receptor to bind the agonist NECA has also been identified. Conformational stabilization of the receptor is thus achieved by reducing basal activity along with modifying the ligand-binding pocket leading to inability to bind agonist. Such markers can be used to screen for stable mutants for structural characterization. Since thermostabilising mutations are not directly transferable across receptors, the yeast based growth assay could serve as a quick and inexpensive way to screen for mutations for a wide range of GPCRs.Open Acces

Loss of constitutive activity is correlated with increased thermostability of the human adenosine A2A receptor

In this note we present an explicit realization of the affine vertex algebra V^cri(gl(1|1)) inside of the tensor product F ⊗ M where F is a fermionic verex algebra and M is a commutative vertex algebra. This immediately gives an alternative description of the center of V^cri(gl(1|1)) as a subalgebra M_0 of M. We reconstruct the Molev-Mukhin formula for the Hilbert-Poincare series of the center of V^cri(gl(1|1)). Moreover, we construct a family of irreducible Vcri(gl(1|1))-modules realized on F and parameterized by χ+, χ- ∈ C((z)). We propose a generalization of V^cri(gl(1|1)) as a critical level version of the super W_{1+∞} vertex algebra

Loss of constitutive activity is correlated with increased thermostability of the human adenosine A2A receptor

BACKGROUND AND PURPOSE: Thermostabilization by mutagenesis is one method which has facilitated the determination of high-resolution structures of the adenosine A(2A) receptor (A(2A)R). Sets of mutations were identified, which both thermostabilized the receptor and resulted in preferential agonist (Rag23 mutant) or antagonist (Rant5 and Rant21) binding forms as assessed by radioligand binding analysis. While the ligand-binding profiles of these mutants are known, the effects these mutations have on receptor activation and downstream signalling are less well characterized. EXPERIMENTAL APPROACH: Here we have investigated the effects of the thermostabilizing mutations on receptor activation using a yeast cell growth assay. The assay employs an engineered Saccharomyces cerevisiae, MMY24, which couples receptor activation to cell growth. KEY RESULTS: Analysis of the receptor activation profile revealed that the wild-type (WT) A(2A)R had considerable constitutive activity. In contrast, the Rag23, Rant5 and Rant21 thermostabilized mutants all exhibited no constitutive activity. While the preferentially antagonist-binding mutants Rant5 and Rant21 showed a complete lack of agonist-induced activity, the Rag23 mutant showed high levels of agonist-induced receptor activity. Further analysis using a mutant intermediate between Rag23 and WT indicated that the loss of constitutive activity observed in the agonist responsive mutants was not due to reduced G-protein coupling. CONCLUSIONS AND IMPLICATIONS: The loss of constitutive activity may be an important feature of these thermostabilized GPCRs. In addition, the constitutively active and agonist-induced active conformations of the A(2A)R are distinct

Molecular evidence of adenosine deaminase linking adenosine A2A receptor and CD26 proteins

Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits

Expression and purification of recombinant G protein-coupled receptors: A review

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques

Compounds which potentiate AMPA receptor and uses thereof in medicine

A compound of formula (I) and salts thereof are provided wherein Y is defined in the specification. Processes for preparation, pharmaceutical compositions, and uses thereof as a medicament, for example in the treatment of a disease or condition mediated by a reduction or imbalance in glutamate receptor function, such as schizophrenia or cognition impairment, are also disclosed

Compounds which potentiate the AMPA receptor and uses thereof in medicine

Compounds of formula (I) and salts thereof are provided: wherein n is 0, 1, 2 or 3; R1 is selected from phenyl and pyridyl, each of which is optionally substituted by one or two groups independently selected from C1-4alkyl and halogen; and R2 is selected from H and CH3 when n is 1 and R2 is H when n is 2 or 3. Processes for preparation, pharmaceutical compositions, and uses thereof as a medicament, for example in the treatment of a disease or condition mediated by a reduction or imbalance in glutamate receptor function, such as schizophrenia or cognition impairment, are also disclosed

Tetrazole compounds as calcium channel blockers

The present invention relates to novel tetrazole compounds; to processes for their preparation; to pharmaceutical compositions containing the compounds; and to the use of the compounds in therapy to treat diseases for which blocking the Cav2.2 calciu

Functional profiles of constructs illustrating the major effects of the A184L mutation.

<p>The A184L mutation alone abolishes constitutive activity (Rag23.29) but this is overcome by the dominant effect of F79A (Rag23.25). The curves (A) are the average of two experiments performed in triplicate. The table (B) shows the pharmacological profile of each mutant characterized. The colours of the curves correspond to those in the table. The data for the WT (pink) are also shown for comparison.</p

Functional profiles of constructs illustrating the major effects of the L272A mutation.

<p>The L272A mutation alone decreases constitutive activity on its own (Rag23.26) but this effect is overcome by the dominant effect of the F79A (Rag23.22). In the case of the combination with the R199A (Rag23.17) mutation there is an almost complete loss of constitutive activity. The curves (A) are the average of two experiments performed in triplicate. The table (B) shows the pharmacological profile of each mutant characterized. The colours of the curves correspond to those in the table. The data for the WT (pink) are also shown for comparison.</p