Modeling and mutational analysis of a putative sodium-binding pocket on the dopamine D2 receptor.
A homology model of the dopamine D2 receptor was constructed based on the crystal structure of rhodopsin. A putative sodium-binding pocket identified in an earlier model (PDB ) was revised. It is now defined by Asn-419 backbone oxygen at the apex of a pyramid and Asp-80, Ser-121, Asn-419, and Ser-420 at each vertex of the planar base. Asn-423 stabilizes this pocket through hydrogen bonds to two of these residues. Highly conserved Asn-52 is positioned near the sodium pocket, where it hydrogen-bonds with Asp-80 and the backbone carbonyl of Ser-420. Mutation of three of these residues, Asn-52 in helix 1, Ser-121 in helix 3, and Ser-420 in helix 7, profoundly altered the properties of the receptor. Mutants in which Asn-52 was replaced with Ala or Leu or Ser-121 was replaced with Leu exhibited no detectable binding of radioligands, although receptor immunoreactivity in the membrane was similar to that in cells expressing the wild-type D2L receptor. A mutant in which Asn-52 was replaced with Gln, preserving hydrogen-bonding capability, was similar to D2L in affinity for ligands and ability to inhibit cAMP accumulation. Mutants in which either Ser-121 or Ser-420 was replaced with Ala or Asn had decreased affinity for agonists (Ser-121), but increased affinity for the antagonists haloperidol and clozapine. Interestingly, the affinity of these Ser-121 and Ser-420 mutants for substituted benzamide antagonists showed little or no dependence on sodium, consistent with our hypothesis that Ser-121 and Ser-420 contribute to the formation of a sodium-binding pocket.
Neve
K A
KA
Portland Veterans Affairs Medical Center and Department of Behavioral Neuroscience, Oregon Health Sciences University, Portland, Oregon, USA. nevek@ohsu.edu
Cumbay
M G
MG
Thompson
K R
KR
Yang
R
R
Buck
D C
DC
Watts
V J
VJ
DuRand
C J
CJ
Teeter
M M
MM
D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics; D001665:Binding Sites; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics; D012964:Sodium / Q000378:metabolism*
D017448:Receptors, Dopamine D2; D012694:Serine; D001216:Asparagine; D012964:Sodium