Phosphorylation of RS1 (RSC1A1) Steers Inhibition of Different Exocytotic Pathways for Glucose Transporter SGLT1 and Nucleoside Transporter CNT1, and an RS1-Derived Peptide Inhibits Glucose Absorption

Abstract

Cellular uptake adapts rapidly to physiologic demands by changing transporter abundance in the plasma membrane. The human gene RSC1A1 codes for a 67-kDa protein named RS1 that has been shown to induce downregulation of the sodium-Dglucose cotransporter 1 (SGLT1) and of the concentrative nucleoside transporter 1 (CNT1) in the plasma membrane by blocking exocytosis at the Golgi. Injecting RS1 fragments into Xenopus laevis oocytes expressing SGLT1 or CNT1 and measuring the expressed uptake of a-methylglucoside or uridine 1 hour later, we identified a RS1 domain (RS1-Reg) containing multiple predicted phosphorylation sites that is responsible for this post-translational downregulation of SGLT1 and CNT1. Dependent on phosphorylation, RS1-Reg blocks the release of SGLT1-containing vesicles from the Golgi in a glucosedependent manner or glucose-independent release of CNT1-containing vesicles. We showed that upregulation of SGLT1 in the small intestine after glucose ingestion is promoted by glucose-dependent disinhibition of the RS1-Reg-blocked exocytotic pathway of SGLT1 between meals. Mimicking phosphorylation of RS1-Reg, we obtained a RS1-Reg variant that downregulates SGLT1 in the brush-border membrane at high luminal glucose concentration. Because RS1 mediates shortterm regulation of various transporters, we propose that the RS1-Reg-navigated transporter release from Golgi represents a basic regulatory mechanism of general importance, which implies the existence of receptor proteins that recognize different phosphorylated forms of RS1-Reg and of complex transporterspecific sorting in the trans-Golgi. RS1-Reg-derived peptides that downregulate SGLT1 at high intracellular glucose concentrations may be used for downregulation of glucose absorption in small intestine, which has been proposed as strategy for treatment of type 2 diabetes.

Sodium glucose cotransporter SGLT1 as a therapeutic target in diabetes mellitus

“…The sweet taste receptor subunit T1R3 and the taste G protein gustducin, expressed in enteroendocrine L-cells, are involved in intestinal sensing of dietary sugar and artificial sweeteners which subsequently up-regulates intestinal SGLT1 mRNA and protein expression [64, 65]. Short-term upregulation of SGLT1 in the brush-border membrane within minutes after glucose ingestion includes the activation and release of SGLT1 containing vesicles from the trans-Golgi network (TGN) [66-70]. The release of SGLT1 containing vesicles from the TGN is steered in a glucose-dependent manner by a highly phosphorylated regulatory domain of the intracellular protein RS1 (gene RSc1A1 ) [70].…”

“…Short-term upregulation of SGLT1 in the brush-border membrane within minutes after glucose ingestion includes the activation and release of SGLT1 containing vesicles from the trans-Golgi network (TGN) [66-70]. The release of SGLT1 containing vesicles from the TGN is steered in a glucose-dependent manner by a highly phosphorylated regulatory domain of the intracellular protein RS1 (gene RSc1A1 ) [70]. An increase in luminal glucose concentrations can also induce GLUT2 translocation to the brush border membrane, however, the quantitative contribution to glucose absorption remains a matter of debate [15, 44, 71-73].…”

“…For example, whereas stimulation of PKC induced short term upregulation of human SGLT1-mediated glucose uptake in oocytes it induced short term downregulation after coexpression of the regulatory protein RS1 which only occurs in mammals [67]. RS1 which is located at the trans-Golgi network (TGN) [68] binds to a receptor at the TGN containing a glucose binding site that blocks the release of SGLT1 containing vesicles from the TGN at low intracellular glucose concentrations [70]. At high intracellular glucose concentrations the RS1-induced blockade of vesicle release is blunted and, as a consequence, vesicle translocation to the plasma membrane is increased leading to a 40-50% increase of SGLT1 expression in the plasma membrane.…”

“…At high intracellular glucose concentrations the RS1-induced blockade of vesicle release is blunted and, as a consequence, vesicle translocation to the plasma membrane is increased leading to a 40-50% increase of SGLT1 expression in the plasma membrane. In RS1 knockout mice the amount of SGLT1 in the plasma membrane is increased at low intracellular glucose and no further increase is observed at high intracellular glucose [70, 89]. Thus, upregulation of SGLT1 expression in small intestine after glucose ingestion is promoted by glucose dependent disinhibition of the RS1-mediated blockade of SGLT1 exocytosis ( Figure 1 ).…”

Expression of JAK3 Sensitive Na+ Coupled Glucose Carrier SGLT1 in Activated Cytotoxic T Lymphocytes

“…For discrimination between those two possibilities SGLT1-expressing Xenopus oocytes were treated with 5 µM brefeldin A, which blocks the insertion of new carrier protein into the cell membrane [74].…”