Constitutively active and dominant-negative G protein coupled receptor mutants

G protein coupled receptors (GPCRs) are thought to exist in equilibrium between inactive and active structures, and agonist binding selectively stabilizes the active conformation. To identify regions of GPCRs that control the transition between the active and inactive states, we identified and analyzed constitutively active and dominant negative mating pheromone receptors of the yeast S. cerevisiae. Constitutively active receptors were generated by sub stituling a proline residue in TMS VI of the alpha-factor receptor (Ste2p) with any other ami no acid. This proline residue in TMS VI may play a conserved role in GPCR activation because it is present in >90% of all GPCRs. and mutation of the analogous proline in another receptor (a-factor receptor) also caused constitutive signalling. To investigate receptor features that stabilize th'1 inactive conformation, we identified a dominant-negative form of the alpha factor receptor, which carried a substitution of a methionine residue in IMS VI. further highlighting the critical role of this region in governing receptor activ ity. This substitution reduced agonist binding affinity 10-fold without affecting cell surface expression levels, as expected for a receptor mutant with impaired signalling activity. Genetic studies suggested the mechanism of the dominant negative effect does not involve G protein sequestration by the mutant receptor. An alternative mechanism may involve receptor oligomerization. which we are currently testing using several experimental strategies.