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.