Conserved polar residues in the transmembrane domain of the human tachykinin NK2 receptor: functional roles and structural implications

Abstract

We have studied the effects of agonist and antagonist binding, agonist-induced activation and agonist-induced desensitization of the human tachykinin NK2 receptor mutated at polar residues Asn-51 [in transmembrane helix 1 (TM1)], Asp-79 (TM2) and Asn-303 (TM7), which are highly conserved in the transmembrane domain in the rhodopsin family of G-protein-coupled receptors. Wild-type and mutant receptors were expressed in both COS-1 cells and Xenopus oocytes. The results show that the N51D mutation results in a receptor which, in contrast with the wild-type receptor, is desensitized by the application of a concentration of 1 microM of the partial agonist GR64349, indicating that the mutant is more sensitive to agonist activation than is the wild-type receptor. In addition, we show that, whereas the D79E mutant displayed activation properties similar to those of the wild-type receptor, the D79N and D79A mutants displayed a severely impaired ability to activate the calcium-dependent chloride current. This suggests that it is the negative charge at Asn-79, rather than the ability of this residue to hydrogen-bond, that is critical for the activity of the receptor. Interestingly, the placement of a negative charge at position 303 could compensate for the removal of the negative charge at position 79, since the double mutant D79N/N303D displayed activation properties similar to those of the wild-type receptor. This suggests that these two residues are functionally coupled, and may even be in close proximity in the three-dimensional structure of the human tachykinin NK2 receptor. A three-dimensional model of the receptor displaying this putative interaction is presented.

Structure of Bovine Rhodopsin in a Trigonal Crystal Form

“…5s-59 For example, the thyrotropin-releasing hormone receptor was completely inactivated by mutation to a non-H-bonding residue at the position of Asp83 or at both positions of Asn55 and Asn302.55-In several receptors an Asp-Asn pair at positions of Asp83 and Asn302 was required for activation of G proteins, but their positions can be interchanged without loss of this activity. [57][58][59] The examples suggest that different H-bonding interactions of these residues are required for stabilising the dark state and for attaining the activated state.…”

Interactions between Conserved Residues in Transmembrane Helices 2 and 7 during Angiotensin AT1 Receptor Activation

“…Sampling was started from the reference point of DT 2 ¼ 0°, DT 7 ¼ 0°(the rhodopsin configuration) and covered most of the points on the grid of DT 2 from )20°to 40°and DT 7 from )20°to 20°. Totally, [25][26][27][28][29][30] configurations of the TM bundle were subjected to energy minimization.…”

“…This was supported by the fact that amino acids corresponding to Asp74 and Asn298 are naturally reversed in some mammalian receptors of the GnRH-R. Interestingly, while reciprocal mutation of these residues partially restored GnRH binding in the mouse GnRH-R (32), no restoration was observed in the same mutants of the rat GnRH-R (33). Subsequent studies suggested the existence of a more extended hydrogen bond network between the conserved residues that correspond to Asn46, Asp74, Asn298, and Tyr302 in the AT 1 receptor in the mouse thyrotropinreleasing hormone receptor (31) and the human tachykinin NK 2 receptor (29). A recent study on the human thyrotropin receptor (51) described a system of interactions between the two residues that involves residues in TM6 and the internal water molecules.…”

Simplified modeling approach suggests structural mechanisms for constitutive activation of the C5a receptor

“…In this study, we also hypothesized that one aspect of receptor activation involves concerted rotations of various TM helices along their long axes. This hypothesis was based on experimental results of numerous site‐directed mutagenesis studies in many GPCRs that were interpreted as indications of TM rotations relative to the ground state structures 30–42. For instance, a recent example of this type of study presented evidence for possible rotation of TM5 in the constitutively active mutant of the angiotensin receptor type 1 43.…”