Specific antibodies against Go isoforms reveal the early expression of the Go2 alpha subunit and appearance of Go1 alpha during neuronal differentiation.
We have previously identified two isoforms of Go alpha in membranes of N1E-115 neuroblastoma cells, using an antibody raised against the purified Go alpha subunit; one isoform of the Go alpha subunit (pI 5.80) is present in undifferentiated cells, whereas a more acidic isoform (pI 5.55) appears during differentiation [J. Neurochem. 54:1310-1320 (1990)]. Recently, the Go alpha gene has been shown to encode, by alternative splicing, two polypeptides, Go1 alpha and Go2 alpha, which differ only in their carboxyl-terminal part. To determine unambiguously whether the two Go alpha subunits detected in neuroblastoma cells were actually the products of different mRNAs, rabbit polyclonal antibodies were generated against synthetic peptides (amino acids 291-302) of both sequences. Specificity of the two affinity-purified antipeptide antibodies was assessed on Western blots by comparing their immunoreactivities with those of other G alpha antibodies. On a blotted mixture of purified brain guanine nucleotide-binding proteins, the anti-alpha o1 and anti-alpha o2 peptide antibodies only recognized the 39-kDa Go alpha subunit. Furthermore, the immunological recognition of brain membranes from 15-day-old mouse fetuses by antipeptide antibodies could be specifically blocked by addition of the corresponding antigen. When membrane proteins from differentiated neuroblastoma cells and mouse fetus brain were blotted after two-dimensional gel electrophoresis, the anti-alpha o1 and anti-alpha o2 peptide antibodies labeled a 39-kDa subunit focused at a pI value of 5.55 or 5.80, respectively. Study of the ontogenesis of both Go alpha subunits revealed the predominance of Go2 alpha in the frontal cortex at day 15 of gestation. Thereafter, there was a progressive decline of the Go2 alpha polypeptide to a very low level, concomitant with an increase in the Go1 alpha protein, which plateaued about 15 days after birth to a level 8 times higher than at gestational day 15. Similarly, on neuroblastoma cells, the Go2 alpha subunit was almost exclusively present in undifferentiated cells, and differentiation induced the appearance of the Go1 alpha subunit, with a reduction in the amount of Go2 alpha polypeptide. Thus, the evolution of the two Go alpha subunits during cell differentiation, unambiguously identified with specific antibodies, suggests that neuronal differentiation is responsible for the on/off switch of the expression of the Go alpha isoforms and indicates that Go1 alpha, rather than Go2 alpha, is involved in neurotransmission.
Rouot
B
B
Centre CNRS-INSERM de Pharmacologie et Endocrinologie, Montpellier, France.
Charpentier
N
N
Chabbert
C
C
Carrette
J
J
Zumbihl
R
R
Bockaert
J
J
Homburger
V
V
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001921:Brain / Q000196:embryology / Q000254:growth & development / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D004247:DNA / Q000235:genetics; D004586:Electrophoresis; D005333:Fetus; D019204:GTP-Binding Proteins / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D015151:Immunoblotting; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000473:pathology / Q000503:physiopathology; D009474:Neurons / Q000502:physiology*; D011817:Rabbits; D014407:Tumor Cells, Cultured
D000906:Antibodies; D046911:Macromolecular Substances; D004247:DNA; D019204:GTP-Binding Proteins