Although the G protein-coupled receptors (GPCRs) share a similar seven-transmembrane domain structure, only a limited number of amino acid residues is conserved in their protein sequences. One of the most highly conserved sequences is the NPxxY motif located at the cytosolic end of helix VII of those GPCRs that belong to the family of the rhodopsin/β-adrenergic-like receptors (Family A). The crystal structure of bovine rhodopsin indicates a hydrophobic interaction between Y7.53 of the NPxxY motif and F7.60 in the helix VIII; moreover, the hydroxyl group of Y7.53 is located close to N2.40 at the cytosolic end of helix II suggesting an interhelical hydrogen bond between helix VII and helix II (Palczewski et al., 2000). Because conservation of a sequence implies an important structural and/or functional role, this motif has been examined by mutagenesis studies in several GPCRs. Interestingly, these mutations affected receptor affinity, signalling, sequestration, and internalization of GPCRs to quite different extents, depending on the receptor under investigation. So far, no general theory could be elaborated that would explain all the observed results. The main purpose of the presented thesis was the investigation of the function of the highly conserved tyrosine (Y7.53) in the NPxxY motif of the human B2 bradykinin receptor (B2R) and its potential interaction partners. Similar to bovine rhodopsin, the B2R also a tyrosine amino acid residue (Y) at position 7.53, a phenylalanine (F) at position 7.60, but a glutamate (E) at position 2.40. The latter, however, is also able to form a hydrogen bond with the hydroxyl group of Y7.53. To study of E2.40↔Y7.53↔F7.60 loci we generated a series of receptor mutants and investigated them with regards to signalling, ligand-inducible phosphorylation, receptor internalization/ sequestration and localization as well as interaction with the Gq/11 protein and G protein-coupled receptor kinases. All experiments were done relatively to the wild-type (wt) receptor. Mutation of F7.60 in helix VIII of the B2R to alanine resulted in a mutant, termed F7.60A. This mutation did not much affect the affinity to its main ligand bradykinin (BK), ligand-inducible phosphorylation, internalization or signalling. In addition, mutant Y7.53F displayed also the properties of the B2R wt. As both point mutants Y7.53F and F7.60A resembled the wild-type phenotype, we assume that the interaction Y7.53↔F7.60could still compensate the absence of an interaction between Y7.53↔E2.40 and vice versa. Furthermore, exchange of Y7.53 to alanine resulted in a mutant, termed Y7.53A, in which both interactions (Y7.53↔F7.60 and Y7.53↔E2.40) were lost. This mutant internalized the ligand [3H]BK almost as rapidly as the B2R wt. However, receptor sequestration of the mutant after stimulation with BK was clearly reduced relatively the B2R wt. Confocal fluorescence microscopy revealed that, in contrast to the B2R, the Y7.53A was predominantly located intracellularly even in the absence of BK. Two-dimensional phosphopeptide analysis showed that the mutant Y7.53A constitutively exhibits a phosphorylation pattern similar to that of the BK-stimulated B2R wt. Ligand-independent Y7.53A internalization was demonstrated by the uptake of rhodamine-labelled antibodies directed to a tag sequence at the N-terminus of the mutated receptor. Co-immunoprecipitation revealed that Y7.53A is pre-coupled to Gq/11 without activating the G protein because the basal accumulation rate of inositol phosphate (IP) was unchanged as compared with B2R wt. We conclude, therefore, that the Y7.53A mutation of B2R induces a semi-active receptor conformation which is prone to ligand-independent phosphorylation and, as a consequence, also to internalization. The mutated receptor binds to, but does not activate, its cognate heterotrimeric G protein Gq/11, thereby limiting the extent of ligand-independent receptor internalization through steric hindrance. Whereas mutation of Y7.53 to phenylalanine (F) or F7.60 to alanine (A) did not much affect ligand-inducible phosphorylation and ligand internalization, the double mutant Y7.53F/F7.60A exhibited a dramatically reduced capability to internalization, most likely caused by the observed resistance to ligand-induced phosphorylation. Co-immunoprecipitation showed that Gq/11 protein was partially pre-coupled to the mutant receptor that probably led to inaccessibility of the mutated receptor for G protein-coupled receptor kinases (GRKs) and, consequently, failure of phosphorylation. However, signal transduction via the G protein Gq/11 was unaffected. The less conservative double mutant Y7.53A/F7.60A was also resistant to phosphorylation and lost its internalization capacity. Moreover, it also showed a strongly reduced IP signal. This double mutant inclined to interaction with GRKs in a ligand-independent manner. But, in contrast to mutant Y7.53A, GRKs phosphorylate the double mutant neither prior nor after stimulation with BK obviously staying bound to the receptor. This may cause the abolished signalling, as the cognate G protein – for sterical reasons – does not have excess to the receptor. To summarize, these data demonstrate that the highly conserved Y7.53 of the NPxxY motif and its potential partners F7.60 and E2.40 play an important role in the interaction of the B2R with the G protein Gq/11 and the GRKs. The binding to/activation of the specific G proteins or recognition by specific kinases requires presentation of a particular spatial distribution of the intracellular docking sites that are normally closed in the inactive state. Thus, our results suggest that the mutation of the Y7.53↔F7.60 locus interferes with a mechanism responsible for stabilizing the receptor in an inactive conformation. Thereby the receptor conformation may be altered and, consequently, the phenotype of the resulting mutants. Finally, it may be concluded that normal receptor activation most likely leads to the disruption of the Y7.53↔F7.60 interaction, too. This information will contribute to a better understanding of the activation mechanism of the B2R; in addition, these data will be very helpful in the creation of the high therapeutic potential B2 receptor agonists and antagonists.
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