O-fucosylation of muscle agrin determines its ability to cluster acetylcholine receptors.
Protein O-fucosyltransferase 1 (Pofut1) transfers fucose to serine or threonine on proteins, including Notch receptors, that contain EGF repeats with a particular consensus sequence. Here we demonstrate that agrin is O-fucosylated in a Pofut1-dependent manner, and that this glycosylation can regulate agrin function. Fucosylation of recombinant C45 agrin, both active (neural, z8) and inactive (muscle, z0) splice forms, was eliminated when agrin was overexpressed in Pofut1-deficient cells or by mutation of a consensus site for Pofut1 fucosylation (serine 1726 in the EGF4 domain). Loss of O-fucosylation caused a gain of function for muscle agrin such that it stimulated AChR clustering and MuSK phosphorylation in cultured myotubes at levels normally only found with the neural splice form. Deletion of Pofut1 in cultured primary myotubes and in adult skeletal muscle increased AChR aggregation. In addition, Pofut1 gene and protein expression and Pofut1 activity of the EGF4 domain of agrin were modulated during neuromuscular development. These data are consistent with a role for Pofut1 in AChR aggregation during synaptogenesis via the regulation of the synaptogenic activity of muscle agrin.
Kim
Mi-Lyang
ML
Center for Gene Therapy, The Research Institute at Nationwide Children's Hospital, Columbus, OH 43205, USA.
Chandrasekharan
Kumaran
K
Glass
Matthew
M
Shi
Shaolin
S
Stahl
Mark C
MC
Kaspar
Brian
B
Stanley
Pamela
P
Martin
Paul T
PT
D018171:Agrin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005643:Fucose / Q000737:chemistry / Q000378:metabolism*; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011940:Receptor Aggregation / Q000502:physiology*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism*; D051880:Receptors, Notch / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013569:Synapses / Q000502:physiology
D018171:Agrin; D034741:RNA, Small Interfering; D011950:Receptors, Cholinergic; D051880:Receptors, Notch; D011994:Recombinant Proteins; D005643:Fucose; D005647:Fucosyltransferases; C105148:polypeptide fucosyltransferase; D020794:Receptor Protein-Tyrosine Kinases
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