ADP-ribosylation factor 6 regulates insulin secretion through plasma membrane phosphatidylinositol 4,5-bisphosphate.
ADP-ribosylation factor 6 (ARF6) is a small GTP-binding protein that regulates peripheral vesicular trafficking and actin cytoskeletal dynamics, and it has been implicated as critical to regulated secretion. Expression of a dominant-inhibitory ARF6 mutant, ARF6(T27N), impaired glucose-, depolarization-, and gamma-thio-GTP-stimulated insulin secretion in the pancreatic beta cell line, MIN6. In response to depolarization, MIN6 cells expressing ARF6(T27N) displayed an unaltered initial fast phase but an impaired subsequent slow phase of insulin secretion. Actin cytoskeletal disassembly with latrunculin A enhanced insulin secretion, whereas stabilization with jasplakinolide inhibited secretion, consistent with the actin cytoskeleton serving as a barrier to exocytosis in these cells. ARF6(T27N) led to a depolarization-dependent reduction in the levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] with a time course that paralleled the inhibition of secretion. Moreover, blockade of PI(4,5)P2-dependent events by expression of a lipid-binding protein resulted in inhibition of depolarization-induced secretion in a manner identical to ARF6(T27N). These results indicate that ARF6 is required to sustain adequate levels of PI(4,5)P2 during periods of increased PI(4,5)P2 metabolism such as regulated secretion.
Howard Hughes Medical Institute, The Cox Institute, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
John T R
JT
Lawrence
Morris J
MJ
Birnbaum
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism / Q000502:physiology*; D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005799:Genes, Dominant; D005822:Genetic Vectors; D005947:Glucose / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D013997:Time Factors
D000089982:ADP-Ribosylation Factor 6; D000199:Actins; D007328:Insulin; D008164:Luminescent Proteins; D010455:Peptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D049452:Green Fluorescent Proteins; D020727:ADP-Ribosylation Factors; C000715727:Arf6 protein, mouse; D005947:Glucose; D002118:Calcium