||In this thesis a combined approach is described to investigate the constitutive activity of G protein protein-coupled receptors (GPCRs) using human adenosine A2B receptors and to evaluate disease-related constitutive GPCR activity as a target for treatment. To this end a yeast expression system together with pharmacological and theoretical receptor models have been applied.
In Chapter 2 the advantages of yeasts as tools to study GPCRs are reviewed. Adapted yeast cells able to communicate with mammalian GPCRs have become available and provide a very convenient system to express mutated receptors. A major advantage of yeast cells over mammalian cells extends from easy culturing conditions to the characteristic of yeast cells to allow entry of only a single plasmid. This latter property in combination with the robust screening assay based on yeast growth makes them an ideal test system to study randomly as well as site-directed mutated receptors. In chapters 3 to 5 this yeast growth assay is the main experimental tool to evaluate the functional properties of random and site-directed mutant receptors.
In chapters 3 and 4 the yeast system is exploited to study inverse agonism of the human adenosine A2B receptor. At first, constitutively active mutant (CAM) human adenosine A2B receptors have been used to discriminate inverse agonists of the adenosine A2B receptor from A2B receptor antagonists. As a result, three inverse agonists ZM241385, DPCPX and MRS1706 were identified and their rank order of efficacy determined. Moreover, an interesting system-dependent phenomenon was noticed, that is the intrinsic activities of the inverse agonists were affected by the level of constitutive activity. It was demonstrated that inverse agonists show the greatest intrinsic activity on receptors displaying a medium level of constitutive activity.
To further investigate the relationship between the effectiveness of an inverse agonist and the level of constitutive activity of the receptor, the two-state receptor model was introduced in both Chapter 3 and Chapter 4. According to this two-state model, both the receptor isomerization constant (L) and the intrinsic efficacy (α) of the inverse agonist determines the sensitivity to detect the intrinsic activity of an inverse agonist which is reflected by the observed experimental window. The biggest experimental window can be achieved on receptors with an L value equaling the reciprocal square root of α. Our experiments show that mutant A2B receptors with an intermediate level of constitutive activity possess the greatest experimental window, whereas mutants with a low level of constitutive activity showed small experimental windows and highly constitutively active mutants did not respond to our tested inverse agonists. Based on these findings we conclude that receptors with intermediate levels of constitutive activity should be the most sensitive screening tools for detecting inverse agonists.
In Chapter 5 the activation of the human adenosine A2B receptor was investigated. To investigate the role of the NxxxNPxxY motif and the potential salt bridge between TM1 (E14) and TM7 (H280) in receptor activation, site-directed mutagenesis was applied to yield 15 mutant A2B receptors. The mutations were selected based on an adenosine A2B receptor model using the structure of bovine rhodopsin as a template. The expression levels of these mutants were determined by western blot analysis and the activation of the receptors was measured in the presence or absence of the following agonists NECA, CPA, CGS21680, IBMECA and LUF5833. None of the mutant receptors displayed constitutive activity. On the contrary, most mutants had a reduced potency and/or efficacy, e.g. mutants N282Q, N282R, N286A, N286Q, N286R, and Y290F showed impaired activation and mutants Y290N, E14H, H280E, E14H/H280E, I61A, I61D and I61K could not be activated by any of the agonists tested. Among all the mutants constructed, only N282R and N286R receptors behaved similarly to the wild-type receptor. Moreover, mutant N286A reduced receptor expression and H280E and E14H/H280E abolished receptor expression. These results suggest an important role for the NxxxNPxxY motif and the potential salt bridge in receptor expression and activation.
The recent publication of the β2-adrenergic receptor structure enabled us to construct a second model of the adenosine A2B receptor. Comparison of the two A2B receptor models based on these two different templates is also described in Chapter 5. The various effects caused by mutations in the NxxxNPxxY motif and the potential salt bridge of different receptors in both our experiments and from literature do suggest that receptor activation is a receptor-specific phenomenon.
In Chapter 6 we provide a theoretical investigation of the treatment of disease-related constitutively active receptor mutations. Comparison of the characteristics of allosteric ligands with traditional orthosteric ligands using a two-state allosteric model predicts that allosteric ligands display a more complicated interaction with a receptor/endogenous ligand pair and are able to cooperatively modify receptor binding and function. As a result allosteric modulators may affect the level of constitutive activity without changing the potency of the endogenous ligand. Thus allosteric modulators may provide advantages over orthosteric ligands in the treatment of diseases caused by constitutively active GPCR mutations.
Finally in Chapter 7, general conclusions about the research described in this thesis are drawn. This is also supplemented by an outlook on some potential aspects of research to be pursued, based upon the application of receptor models, pharmacology models and functional receptor assays.