Arginine 199 and leucine 208 have key roles in the control of adenosine A2A receptor signalling function

Abstract

One successful approach to obtaining high-resolution crystal structures of G-protein coupled receptors is the introduction of thermostabilising mutations within the receptor. This technique allows the generation of receptor constructs stabilised into different conformations suitable for structural studies. Previously, we functionally characterised a number of mutants of the adenosine A2A receptor, thermostabilised either in an agonist or antagonist conformation, using a yeast cell growth assay and demonstrated that there is a correlation between thermostability and loss of constitutive activity. Here we report the functional characterisation of 30 mutants intermediate between the Rag23 (agonist conformation mutant) and the wild-type receptor using the same yeast signalling assay with the aim of gaining greater insight into the role individual amino acids have in receptor function. The data showed that R199 and L208 have important roles in receptor function; substituting either of these residues for alanine abolishes constitutive activity. In addition, the R199A mutation markedly reduces receptor potency while L208A reduces receptor efficacy. A184L and L272A mutations also reduce constitutive activity and potency although to a lesser extent than the R199A and L208A. In contrast, the F79A mutation increases constitutive activity, potency and efficacy of the receptor. These findings shed new light on the role individual residues have on stability of the receptor and also provide some clues as to the regions of the protein responsible for constitutive activity. Furthermore, the available adenosine A2A receptor structures have allowed us to put our findings into a structural context.

Figure 1. Functional profiles of constructs illustrating the major effects of the F79A mutation.

The F79A mutation increases constitutive activity, efficacy and potency, both alone (Rag23.30) and in combination with other mutations (Rag23.13, Rag23.14, Rag23.22, Rag23.23, Rag23.24, Rag23.25, Rag23.27, Rag23.28 and Rag23.30). The curves (A and B) are the average of two experiments performed in triplicate. The table (C) shows the pharmacological profile of each mutant characterized. The colours of the curves correspond to those in the table. The data for the WT and Rag23 constructs (pink) are also shown for comparison.

Figure 2. Functional profiles of constructs illustrating the major effects of the R199A mutation.

The R199A mutation reduces constitutive activity and potency, both alone (Rag23.28) and in combination with other mutations (Rag23.6, Rag23.17, Rag23.21, Rag23.24 and Rag23.28). 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.

Figure 3. Functional profiles of constructs illustrating the major effects of the L208A mutation.

The L208A mutation reduces constitutive activity and efficacy, both alone (Rag23.27) and in combination with other mutations (Rag23.1, Rag23.2, Rag23.3, Rag23.4, Rag23.5, Rag23.10, Rag23.14, Rag23.20 and Rag23.27). The curves are the average of two experiments performed in triplicate. The table (C) 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.

Figure 4. Functional profiles of constructs illustrating the major effects of the A184L mutation.

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.

Figure 5. Functional profiles of constructs illustrating the major effects of the L272A mutation.

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.

Figure 6

Position of the F79 (red), R199A and L208 (magenta), A184 and L272 (blue) residues in the high-resolution crystal structure of the A_2AR GL31 thermostable mutant (PDB accession code: 2YDV) (A). F79 is located on TM3 (B) while R199 and L208 are both located at the cytoplasmic end of TM5 (C) and A184 and L272 are located at the extracellular end of TM5 and TM7 respectively (D).