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

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