Mutational analysis of the role of GXXXG motif in the function of human organic anion transporter 1 (hOAT1)

摘要

Human organic anion transporter hOAT1 plays a critical role in the body disposition of environmental toxins and clinically important drugs including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. hOAT1 has two GXXXG motifs in its transmembrane domains 2 and 5, a motif linked to the protein processing and oligomerization of other proteins. In the current study, we substituted glycine of these GXXXG motifs with alanine and evaluated the effect of such mutations on the expression and function of hOAT1. Mutations of GXXXG motif in the transmembrane domain 2 resulted in mutants G144A and G148A, both of which had no transport activity due to complete loss in the surface and total cell expression of the transporter protein. Treatment of G144A- and G148A-expressing cells with proteasomal inhibitor resulted in the recovery of ER-resident immature form of hOAT1, but not its surface-resident mature form, whereas treatment of these cells with lysosomal inhibitor had no effect on the expression of the mutant transporters. Mutations of GXXXG motif in the transmembrane domain 5 resulted in mutants G223A and G227A, among which only G227 had dramatic reduction of transport activity due to dramatic loss in the surface and total cell expression of the transporter. The reduction in the surface expression of G227 was consistent with the decrease in maximum transport velocity Vmax. Treatment of G227A-expressing cells with proteasomal inhibitor or lysosomal inhibitor resulted in partial recovery of both the immature form and the mature form of hOAT1 in the total cell extracts. However, such partial recovery of the mature form in total cell extracts did not lead to the partial recovery of surface expression and function of the transporter. Our data suggest that the GXXXG motifs in transmembrane domains 2 and 5 play critical roles in the stability of hOAT1.