Abstract

Introduction

Signals for Polarized Sorting

Regulation of Transporter Function by Endo and Exocytosis

Conclusions

References

Discussion
Board

The cell surface membranes of epithelial cells are divided into two distinc t domains characterized by distinct protein compositions, reflecting their different roles in vectorial solute transport. We are interested in the molecular signals and cellular machinery involved in generating this polarity. Membrane proteins whose distributions are restricted to one or the other cell surface domains of polarized cells must contain within their structure information which specifies their appropriate subcellular destination. Our studies of these 'sorting signals' make use of families of transport proteins, each of whose members are sorted to different surfaces despite extensive homology. The transport systems involved in neurotransmitter re-uptake are highly homologous but differentially sorted. We are generating molecular chimeras composed of complementary portions of related transporters and expressing them by transfection in polarized epithelial cells. This approach has allowed us to identify segments of these molecules which contribute to their sorting behaviors as well as to their physiologic properties. Our studies of neurotransmitter re-uptake systems have demonstrated that the four members of the highly homologous GABA transporter gene family are differentially sorted in epithelial cells and in neurons. We find that the GAT-1 and GAT-3 isoforms, which are restricted to axons when expressed endogenously or by transfection in neurons, are sorted to the apical membranes of epithelial cells. The GAT-2 and betaine transporters, which are 50-67% identical to GAT-1 and GAT-3, behave as basolateral proteins in epithelia and are restricted to dendrites when we express them in neurons. Production of chimeric and deletion constructs has allowed us to identify a 32 amino acid sequence in the C-terminal tail of these transporters which we believe manifests targeting information. Future studies should allow us to characterize this sequence and to identify the epithelial and neuronal proteins which interact with it.

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Presentation Number SAmuth0302
Keywords: Polarity, Sorting, Transporter, Neurotransmitter, GABA