Conserved Helix 7 Tyrosine Acts as a Multistate Conformational Switch in the 5HT2C Receptor

Abstract

Studies in many rhodopsin-like G-protein-coupled receptors are providing a general scheme of the structural processes underlying receptor activation. Microdomains in several receptors have been identified that appear to function as activation switches. However, evidence is emerging that these receptor proteins exist in multiple conformational states. To study the molecular control of this switching process, we investigated the function of a microdomain involving the conserved helix 7 tyrosine in the serotonin 5HT2C receptor. This tyrosine of the NPXXY motif was substituted for all naturally occurring amino acids. Three distinct constitutively active receptor phenotypes were found: moderate, high, and "locked-on" constitutive activity. In contrast to the activity of the other receptor mutants, the high basal signaling of the locked-on Y7.53N mutant was neither increased by agonists nor decreased by inverse agonists. The Y7.53F mutant was uncoupled. Computational modeling based on the rhodopsin crystal structure suggested that Y7.53 interacts with the conserved aromatic ring at position 7.60 in the recently identified helix 8 domain. This provided a basis for seeking revertant mutations to correct the defective function of the Y7.53F receptor. When the Y7.53F receptor was mutated at position 7.60, the wild-type phenotype was restored. These results suggest that Y7.53 and Y7.60 contribute to a common functional microdomain connecting helices 7 and 8 that influences the switching of the 5HT2C receptor among multiple active and inactive conformations.

Role of the conserved NPxxY(x) 5,6 F motif in the rhodopsin ground state and during activation

“…Several lines of evidence suggest a structural rearrangement of H8 after receptor activation (15,(26)(27)(28)(29)(30)(31)(32). Here, we showed that the release of the constraint on the NPxxY(x) 5,6 F region imposed by the Y306-F313 interaction occurs during the Meta I͞Meta II transition as unveiled by UV-visible spectroscopy.…”

“…Mutations in the NPxxY(x) 5,6 F motif affect receptor expression, ligand affinity, receptor sequestration, heterotrimeric G protein coupling, and association with the small G proteins ARF and RhoA (for example, see refs. [13][14][15][16].…”

Mutation of Tyrosine in the Conserved NPXXY Sequence Leads to Constitutive Phosphorylation and Internalization, but Not Signaling, of the Human B2 Bradykinin Receptor

“…Because conservation of the pentapeptide sequence implies an important structural or functional role, this motif has been examined by mutagenesis studies in several GPCRs. These mutations affected receptor affinity (5,7,8), signaling (3,5,6,9), sequestration (4, 13), and internalization of GPCRs (12) but to quite different extents, depending on the receptor under study. This may, in part, be reflective of the microenvironment surrounding this motif in the respective receptor sequence, the assays applied, or the cells used for ex- pression.…”

“…One of the most highly conserved motifs, together with the E/DRY sequence at the cytosolic end of transmembrane domain-3, is the NPXXY sequence (where X usually represents a hydrophobic residue and N is rarely exchanged against D) located at the C terminus of transmembrane domain-7. The results of these mutagenesis studies implicate this motif in the signaling, sequestration, and internalization of many GPCRs (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13).…”

An Activation Switch in the Rhodopsin Family of G Protein-coupled Receptors

“…Agonist binding or constitutively active mutations disrupt a series of constraining intramolecular interactions that keep the receptor in the inactive conformation. Recently identified constraints within the transmembrane domain are i) the conformational transition of Trp of the CWxP motif in TM 6 (11,12), ii) the interaction of Asn of the NPxxY motif in TM 7 with either residues located in TM 6 (13) or the backbone of TM 6 through a water molecule (14), iii) the ionic lock between Arg of the highly conserved (D/E)RY motif in TM 3 and a partly conserved Asp/Glu residue in the cytoplasmic end of TM 6 (15), and iv) the aromatic-aromatic interaction between Tyr of the NPxxY motif in TM 7 and an aromatic residue located in HX 8 (16,17). Release of these constraints induce rigid body motions of several if not all transmembrane helices (18,19).…”