Mutagenesis of ser41 to ala inhibits the association of GAP-43 with the membrane skeleton of GAP-43-deficient PC12B cells: effects on cell adhesion and the composition of neurite cytoskeleton and membrane.
To investigate the molecular basis for GAP-43 function in axon outgrowth, we produced a mutant, GAP-43 (Ala41), whose interaction with calmodulin in vitro was unaffected by increasing Ca2+ concentrations, and stably transfected it into GAP-43-deficient PC12B cells. Several lines that expressed wild-type or mutant protein at levels that resembled endogenous GAP-43 expression in PC12 controls were subcloned and characterized. GAP-43 (Ala41) was significantly more extractable with Nonidet P-40 and less tightly associated with the membrane skeleton than the wild-type protein. Furthermore, GAP-43 (Ala41) expression by PC12B cells profoundly affected their phenotype: First, observation of living cells using video-enhanced microscopy revealed irregular plasma membranes with numerous blebs and protrusions and neurites that appeared thin and varicose. Second, both the cells' ability to remain attached to laminin substrates and the amount of alpha 1 beta 1 integrin expressed on the cell surface was significantly decreased. Finally, peripherin transport, which is abnormal in PC12B cells, could be rescued by transfection of wild-type GAP-43 but not the GAP-43 (Ala41) mutant. The phenotypic abnormalities resemble other cell types in which membrane skeleton/plasma membrane interactions have been functionally decoupled, and our results are consistent with the notion that these interactions may be abnormal in GAP-43 (Ala41)-expressing PC12B cells, either as a direct consequence of the mutation or arising secondarily to the altered availability of calmodulin in the growing neurite.
Meiri
K F
KF
Department of Pharmacology, SUNY Health Science Center, Syracuse, New York 13210, USA.
Hammang
J P
JP
Dent
E W
EW
Baetge
E E
EE
D000409:Alanine / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D019922:GAP-43 Protein; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D016023:Integrins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D018715:Microscopy, Video; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism* / Q000648:ultrastructure; D016900:Neurofilament Proteins / Q000235:genetics* / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D012694:Serine / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection
D002147:Calmodulin; D019922:GAP-43 Protein; D016023:Integrins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D012694:Serine; D000409:Alanine; D002118:Calcium