The role of proline residues in the structure and function of human MT2 melatonin receptor.
Melatonin functions as an essential regulator of various physiological processes in all vertebrate species. In mammals, two G protein-coupled melatonin receptors (GPCR) mediate some melatonin's actions: MT1 and MT2. Transmembrane domains (TM) of most GPCRs contain a set of highly conserved proline residues that presumably play important structural and functional roles. As TM segments of MT2 receptor display several interesting differences in expression of specific proline residues compared to other rhodopsin-like receptors (rGPCRs), we investigated the role of proline residues in the structure and function of this receptor. All prolines in TM segments of MT2 receptor were individually replaced with alanine and/or glycine. In addition, the unusual NAxxY motif located in TM7 was mutated to generate highly conserved NPxxY motif found in the majority of rGPCR proteins. Following transient expression in CHO-K1 cells, binding properties of the mutant receptors and their ability to transduce signals were analyzed using (125)I-mel- and [(35)S]GTPgammaS-binding assays, respectively. The impact of the performed mutations on the receptor structure was assessed by molecular dynamic simulations of MT2 receptors embedded in the fully hydrated phospholipid bilayer. Our results indicate that residues P174, P212 and P266 are important for the ligand binding and/or signaling of the human MT2 receptor. We also show that changes within the unusual NAxxY sequence in the TM7 (mutations A305P and A305V) produce defective MT2 receptors indicating an important role of this motif in the function of melatonin receptors.
Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Petr
P
Mazna
Lenka
L
Grycova
Ales
A
Balik
Hana
H
Zemkova
Eliska
E
Friedlova
Veronika
V
Obsilova
Tomas
T
Obsil
Jan
J
Teisinger
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D003198:Computer Simulation; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007150:Immunohistochemistry; D007457:Iodine Radioisotopes; D008550:Melatonin / Q000378:metabolism; D008565:Membrane Proteins; D018613:Microscopy, Confocal; D008958:Models, Molecular; D009154:Mutation; D011392:Proline / Q000502:physiology*; D017434:Protein Structure, Tertiary; D044123:Receptor, Melatonin, MT2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D044123:Receptor, Melatonin, MT2; D013462:Sulfur Radioisotopes; D011392:Proline; D008550:Melatonin