Characterization of the Major Bovine Brain Go α Isoforms

Abstract

G o is the major G protein in bovine brain, with at least three isoforms, G oA , G oB , and G oC . Whereas ␣ oA and ␣ oB arise from a single G o ␣ gene as alternatively spliced mRNAs, ␣ oA and ␣ oC are thought to differ by covalent modification. To test the hypothesis that ␣ oA and ␣ oC have different N-terminal lipid modifications, proteolytic fragments of ␣ o isoforms were immunoprecipitated with an N terminus-specific antibody and analyzed by matrix-assisted laser desorption ionization mass spectrometry. The major masses observed in immunoprecipitates were the same for all three ␣ o isoforms and corresponded to the predicted mass of a myristoylated N-terminal fragment. Structural differences between ␣ oA and ␣ oC were also compared before and after limited tryptic proteolysis using SDS-polyacrylamide gel electrophoresis containing 6 M urea. Based upon the ␣ o subunit fragments produced under activating and nonactivating conditions, differences between ␣ oA and ␣ oC were localized to a C-terminal fragment of the protein. This region, involved in receptor and effector interactions, implies divergent signaling roles for these two ␣ o proteins. Finally, the structural difference between ␣ oA and ␣ oC is associated with a difference of at most 2 daltons based upon measurements by electrospay ionization mass spectrometry.G proteins, composed of an ␣ subunit bound to a ␤␥ dimer, mediate the effects of many extracellular ligands that act on specific cell surface receptors (1). G o is the major brain G protein, comprising up to 1% of particulate protein in bovine brain (2, 3). The exact function of G o is still unclear, but it has been implicated in the regulation of voltage-gated calcium channels (4 -6); the activation of the mitogen-activated protein kinase pathway (7); the development of neuronal growth cones (8), where it is highly concentrated (9); and the regulation of vesicle trafficking (10 -13).The role of the ␣ o subunit on the level of the whole animal has also been studied. In Caenorhabditis elegans, the ␣ o protein influences behaviors such as locomotion and reproduction (14,15). Mice lacking the ␣ o gene are afflicted with tremors and seizures (16,17) and show loss of motor control and a propensity to run continuously in circles in a counterclockwise direction (17). In both studies with mice deficient in ␣ o , life span was significantly reduced. Although the precise function of ␣ o in neuronal tissue remains to be defined, in the knockout mice, regulation of Ca 2ϩ channels by opioid receptors in dorsal root ganglion cells is altered (17), as is regulation of L-type calcium channels in heart (16). Thus, the expression of ␣ o is clearly required for normal neuronal function.One gene codes for the ␣ o subunit, which gives rise in brain to multiple splice variants with two different coding sequences contained in mRNAs ␣ o1 and ␣ o2 (18 -20). Based upon protein characterization, however, there are at least four ␣ o isoforms in bovine brain, ␣ oA , ␣ oB , ␣ oC , and ␣ oD (21-23). Three of the isoforms, ␣ oA ,...

Differentiation of Human T Cells Alters Their Repertoire of G Protein α-Subunits

“…derived from posttranslational modifications of G␣ oA and G␣ oB , respectively (45). Within the nervous system, G␣ o activates neuronal Ca 2ϩ and K ϩ channels (46 -50).…”

Specialized Cilia in Mammalian Sensory Systems

“…Patients with ciliopathies affecting the inner ear are often deaf and/or exhibit balance difficulties [ 22 ]. Patients with an affected olfactory epithelium often show a complete loss of smell (anosmia) [ 23 , 24 ], and patients with retinal ciliopathies often become blind (retinal degeneration) [ 25 , 26 , 27 ]. Impairment of more than one sensory organ results, for instance, in Bardet-Biedl [ 28 , 29 ] or Usher syndrome [ 30 , 31 ], two autosomal recessive genetic diseases ( Table 1 ).…”

Osmoregulated periplasmic glucans in Proteobacteria

“…The ¢rst report of OPG structures is probably the description by McIntire et al [2], in 1942, of cyclic L,1-2 glucans found in culture ¢ltrates of a crown-gall organism (Agrobacterium tumefaciens). In this work, the glucans were essentially thought as a particular sub-class of exopolysaccharides.…”