Role of tryptophan-388 of GLUT1 glucose transporter in glucose-transport activity and photoaffinity-labelling with forskolin

H. Katagiri, T. Asano, H. Ishihara, J. L. Lin, K. Inukai, M. F. Shanahan, K. Tsukuda, M. Kikuchi, Y. Yazaki, Y. Oka

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Abstract

GLUT1 glucose-transporter cDNA was modified to substitute leucine for Trp-388 and transfected into Chinese hamster ovary cells using the expression vector termed pMTHneo. This tryptophan residue is conserved among most of the facilitative glucose-transporter isoforms and has been proposed to be the photolabelling site of forskolin, a competitive inhibitor of glucose transport. In addition, this residue is located on membrane-spanning helix 10 which is suggested to contain the dynamic segment of the transporter. The mutated glucose transporter was expressed and inserted into the plasma membrane in a fashion similar to the wild-type. Unexpectedly, this mutation did not abolish photolabelling with forskolin. However, the mutation induced a marked decrease in 2-deoxyglucose uptake with a 4-fold decrease in turnover number and a 1.25-fold increase in K(m) compared with the wild-type GLUT1. A similar decrease in zero trans influx activity was also observed for 3-O-methylglucose. In contrast, no apparent decrease was observed in zero trans efflux activity for 3-O-methylglucose. The mutation decreased the turnover number of the glucose transporter in equilibrium exchange influx for 3-O-methylglucose by 33% without any change in K(m). These results indicate that (1) Trp-388 is not the photolabelling site for forskolin, if we assume that the labelling occurs at a single site and (2) Trp-388 is more likely to be involved in interconversion between the inward-facing and outward-facing conformers of GLUT1 than binding of glucose, and thus, substitution of leucine for Trp-388 in this dynamic segment would decrease the rate of alternating conformation, which would preferentially affect the influx activity.

Original languageEnglish
Pages (from-to)861-867
Number of pages7
JournalBiochemical Journal
Volume291
Issue number3
DOIs
Publication statusPublished - 1993 Jan 1
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Katagiri, H., Asano, T., Ishihara, H., Lin, J. L., Inukai, K., Shanahan, M. F., Tsukuda, K., Kikuchi, M., Yazaki, Y., & Oka, Y. (1993). Role of tryptophan-388 of GLUT1 glucose transporter in glucose-transport activity and photoaffinity-labelling with forskolin. Biochemical Journal, 291(3), 861-867. https://doi.org/10.1042/bj2910861