Effect of N-glycosylation on ligand binding affinity of rat V1a vasopressin receptor.
A rat Vla vasopressin (rVla) receptor has two putative N-glycosylation sites at 14th and 27th amino acid asparagine in the extracellular N-terminus. In the present study, we examined the possible roles of N-glycosylation of the N-terminus in the receptor function. Three point mutants for deglycosylated rVla receptor were generated in which the 14th and/or the 27th asparagine was replaced with glutamine, namely N14Q, N27Q, and N14:27Q, each tagged with an enhanced green fluorescent protein (EGFP) at their C-termini, and transfected to COS-7 or HEK292 cells. The two single mutants and a double mutant have progressively smaller molecular mass compared to the wild-type receptor as determined by immunoblot analysis, indicating that the two sites are effectively glycosylated in vivo. The maximal ligand binding capacities of three mutant receptors were comparable to that of wild-type (17.02 +/- 1.32 pmol/g protein) with modest changes in ligand binding affinities: N27Q and N14:27Q had decreased binding affinities compared to N14Q and wild-type receptors. The reduced binding affinities of the deglycosylated mutants are not likely due to the impaired intracellular transport since their traffickings were indistinguishable from one another. Taken together, these results suggest that the N-glycosylation at the two sites of the N-terminus of rV1a receptor minimally affects the surface expression and trafficking of the receptor.
Lee
K H
KH
Department of Biochemistry, College of Medicine, Hanyang University, Seoul, Korea.
Ahn
J I
JI
Yu
D H
DH
Jeong
H S
HS
Lee
S H
SH
Kim
K S
KS
Chung
I Y
IY
Kim
J H
JH
Lee
Y S
YS
D000818:Animals; D001216:Asparagine / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D006031:Glycosylation; D017354:Point Mutation; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D017483:Receptors, Vasopressin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014667:Vasopressins / Q000378:metabolism
D017483:Receptors, Vasopressin; D014667:Vasopressins; D001216:Asparagine