An amino-terminal domain of the growth-associated protein GAP-43 mediates its effects on filopodial formation and cell spreading.
GAP-43 is a neuronal protein that is believed to be important to neuronal growth and nerve terminal plasticity. It is enriched on the inner surface of growth cone membranes, a localization that may depend upon palmitoylation of Cys3 and Cys4. It is a major substrate for protein kinase C, which phosphorylates Ser41. Isolated GAP-43 can bind to actin and to calmodulin, and can activate the heterotrimeric GTP-binding proteins, G(o) and Gi. A peptide consisting of the GAP-43 sequence 39-55 binds calmodulin, and an amino-terminal GAP-43 (1-10) peptide activates G(o), suggesting that these stretches may be functional domains of the intact protein. When expressed in non-neuronal cells, GAP-43 enhances filopodial extension and has effects upon cell spreading. We have examined the effects of various GAP-43 domains upon this assay, by expression of GAP-43, GAP-43 mutant proteins, and GAP-43-CAT fusion proteins in COS-7 cells. We find that the amino terminus (Met-Leu-Cys-Cys-Met-Arg-Arg-Thr-Lys-Gln) is an important contributor to these effects on cell shape. A GAP-43 protein mutant in Cys3 and Cys4 does not bind to the membrane, and is inactive. Mutants in Arg6 or Lys9 also are inactive, although they remain localized to particulate fractions; Arg7 mutants are active. A chimeric gene consisting of GAP-43 (1-10) fused to chloramphenicol acetyl transferase (CAT) also causes cell shape changes. As for GAP-43, the effects of this fusion protein are abolished by mutations of Cys3, Cys4, Arg6 or Lys9, but not by mutation of Arg7. Therefore, the cell surface activity of transfected GAP-43 depends upon its amino terminus, although other domains may regulate it in this regard. Since the amino-terminal domain includes the peptide stretch known to be capable of activating G(o) and Gi, we examined the effect of GAP-43 on a Gi-regulated second messenger system, the inhibition of cAMP production in A431 cells. A431 cells stably transfected with GAP-43 spread less well than do controls. In addition, they evidence decreased levels of forskolin-stimulated cAMP, consistent with chronic stimulation of Gi. Stimulation of adenylate cyclase by isoproterenol reverses the GAP-43-induced changes in cell shape. This suggests that G protein stimulation is involved in GAP-43 effects upon cell shape.
Strittmatter
S M
SM
Developmental Biology Laboratory, Massachusetts General Hospital, Charlestown.
Valenzuela
D
D
Fishman
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure*; D002465:Cell Movement / Q000502:physiology*; D002522:Chlorocebus aethiops; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D019922:GAP-43 Protein; D015870:Gene Expression; D005822:Genetic Vectors; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000502:physiology; D010957:Plasmids; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014162:Transfection; D014407:Tumor Cells, Cultured
D019922:GAP-43 Protein; D006133:Growth Substances; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D005576:Colforsin; D000242:Cyclic AMP