An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor.
We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of approximately 14 and approximately 10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of approximately 13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.
Urizar
Eneko
E
Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Université Libre de Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Bruxelles, Belgium.
Claeysen
Sylvie
S
Deupí
Xavier
X
Govaerts
Cedric
C
Costagliola
Sabine
S
Vassart
Gilbert
G
Pardo
Leonardo
L
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D019556:COS Cells; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006860:Hydrogen Bonding; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000378:metabolism*; D012243:Rhodopsin / Q000737:chemistry*; D012984:Software; D055672:Static Electricity; D013912:Threonine / Q000737:chemistry; D014162:Transfection
D043562:Receptors, G-Protein-Coupled; D011989:Receptors, Thyrotropin; D013912:Threonine; D001224:Aspartic Acid; D001216:Asparagine; D012243:Rhodopsin; D000242:Cyclic AMP; D007930:Leucine; D000409:Alanine