Glucose activates mitogen-activated protein kinase (extracellular signal-regulated kinase) through proline-rich tyrosine kinase-2 and the Glut1 glucose transporter.

Glucose serves as both a nutrient and regulator of physiological and pathological processes. Presently, we found that glucose and certain sugars rapidly activated extracellular signal-regulated kinase (ERK) by a mechanism that was: (a) independent of glucose uptake/metabolism and protein kinase C but nevertheless cytochalasin B-inhibitable; (b) dependent upon proline-rich tyrosine kinase-2 (PYK2), GRB2, SOS, RAS, RAF, and MEK1; and (c) amplified by overexpression of the Glut1, but not Glut2, Glut3, or Glut4, glucose transporter. This amplifying effect was independent of glucose uptake but dependent on residues 463-468, IASGFR, in the Glut1 C terminus. Accordingly, glucose effects on ERK were amplified by expression of Glut4/Glut1 or Glut2/Glut1 chimeras containing IASGFR but not by Glut1/Glut4 or Glut1/Glut2 chimeras lacking these residues. Also, deletion of Glut1 residues 469-492 was without effect, but mutations involving serine 465 or arginine 468 yielded dominant-negative forms that inhibited glucose-dependent ERK activation. Glucose stimulated the phosphorylation of tyrosine residues 402 and 881 in PYK2 and binding of PYK2 to Myc-Glut1. Our findings suggest that: (a) glucose activates the GRB2/SOS/RAS/RAF/MEK1/ERK pathway by a mechanism that requires PYK2 and residues 463-468, IASGFR, in the Glut1 C terminus and (b) Glut1 serves as a sensor, transducer, and amplifier for glucose signaling to PYK2 and ERK.

Regulation of Cl-/ HCO3- exchange by cystic fibrosis transmembrane conductance regulator expressed in NIH 3T3 and HEK 293 cells.

A central function of cystic fibrosis transmembrane conductance regulator (CFTR)-expressing tissues is the secretion of fluid containing 100-140 mM HCO3-. High levels of HCO3- maintain secreted proteins such as mucins (all tissues) and digestive enzymes (pancreas) in a soluble and/or inactive state. HCO3- secretion is impaired in CF in all CFTR-expressing, HCO3--secreting tissues examined. The mechanism responsible for this critical problem in CF is unknown. Since a major component of HCO3- secretion in CFTR-expressing cells is mediated by the action of a Cl-/HCO3- exchanger (AE), in the present work we examined the regulation of AE activity by CFTR. In NIH 3T3 cells stably transfected with wild type CFTR and in HEK 293 cells expressing WT and several mutant CFTR, activation of CFTR by cAMP stimulated AE activity. Pharmacological and mutagenesis studies indicated that expression of CFTR in the plasma membrane, but not the Cl- conductive function of CFTR was required for activation of AE. Furthermore, mutations in NBD2 altered regulation of AE activity by CFTR independent of their effect on Cl- channel activity. At very high expression levels CFTR modified the sensitivity of AE to 4,4'-diisothiocyanatostilbene-2, 2'-disulfonate. The novel finding of regulation of Cl-/HCO3- exchange by CFTR reported here may have important physiological implications and explain, at least in part, the impaired HCO3- secretion in CF.

Structural domains that contribute to substrate specificity in facilitated glucose transporters are distinct from those involved in kinetic function: studies with GLUT-1/GLUT-2 chimeras.

GLUT-2 differs from other members of the facilitated glucose transporter family because it transports a wider range of substrates and exhibits a higher Km for transport of glucose analogs such as 2-deoxyglucose (2-DOG). In order to investigate the structural determinants of the unique substrate specificity and kinetic function of GLUT-2, recombinant adenoviruses were used to express native, mutant, and chimeric glucose transporters in the kidney cell line CV-1, yielding the following key observations. (1) A chimera consisting of GLUT-1 with the C-terminal tail of GLUT-2 had a Km for 2-DOG of 9.9 +/- 1.5 that was intermediate between that of native GLUT-1 (3.7 +/- 0.4) and native GLUT-2 (26.3 +/- 3.3). In contrast to the effect of the GLUT-2 C terminus on Km for 2-DOG, this substitution did not confer enhanced uptake of three alternative substrates (fructose, arabinose, or streptozotocin) which are transported efficiently by native GLUT-2 but not by GLUT-1. (2) A chimera consisting of GLUT-2 with the N-terminal 87 amino acids of GLUT-1 exhibited no change in Km for 2-DOG relative to native GLUT-2 but exhibited a significant reduction in capacity for transport of the three alternative substrates. (3) Mutation of asparagine 62 in GLUT-2 to glutamine produced a transporter lacking its N-linked oligosaccharide that exhibited a 2.5-fold increase in Km for 2-DOG but equally efficient transport of the three alternative substrates relative to native GLUT-2. These data provide insight into structural domains that affect substrate specificity in facilitated glucose transporters and demonstrate that they are distinct from elements involved in glucose transport kinetics.