The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H-2 receptor

In previous studies we showed that the wild-type histamine H-2 receptor sta bly expressed in Chinese hamster ovary cells is constitutively active. Beca use constitutive activity of the H-2 receptor is already found at low expre ssion levels (300 fmol/mg protein) this receptor is a relatively unique mem ber of the G-protein-coupled receptor (GPCR) family and a useful tool for s tudying GPCR activation. In this study the role of the highly conserved DRY motif in activation of the H-2 receptor was investigated. Mutation of the aspartate 115 residue in this motif resulted in H-2 receptors with high con stitutive activity, increased agonist affinity, and increased signaling pro perties. In addition, the mutant receptors were shown to be highly structur ally instable. Mutation of the arginine 116 residue in the DRY motif result ed also in a highly structurally instable receptor; expression of the recep tor could only be detected after stabilization with either an agonist or in verse agonist. Moreover, the agonist affinity at the Arg-116 mutant recepto rs was increased, whereas the signal transduction properties of these recep tors were decreased. We conclude that the Arg-116 mutant receptors can adop t an active conformation but have a decreased ability to couple to or activ ate the G(s)-protein. This study examines the pivotal role of the aspartate and arginine residues of the DRY motif in GPCR function. Disruption of rec eptor stabilizing constraints by mutation in the DRY motif leads to the for mation of active GPCR conformations, but concomitantly to GPCR instability.