Signal transduction by guanine nucleotide-binding proteins.
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Biomedical subjects
M Brann
Publications and source records attributed to M Brann.
Detection of multiple forms of Gi alpha in HL60 cells.
Comparison of cDNA sequences from multiple sources predicts a genus of highly homologous but structurally distinct G protein alpha-subunits, designated as Gi alpha, that may include the alpha-subunit of the functionally defined adenylate cyclase inhibitory G protein. Using specific oligonucleotide probes on Northern blots, we show that Gi alpha-2 and Gi alpha-3, but not Gi alpha-1, are expressed in HL60 cells. Antisera raised against synthetic peptides from regions predicted to be conserved (AS6) and divergent (LE3) among Gi alpha subtypes bind to a 40 kDa protein in Western blots of HL60 membranes. AS6 identifies an additional protein at 41 kDa. Thus, Northern blot and immunoblot results show that at least two Gi alpha subtypes, a 40 kDa protein likely to correspond to Gi alpha-2 and a 41 kDa protein possibly representing Gi alpha-3, may be expressed in a single cell type.
Amino Acid Sequence↗
The use of [3H](-)-DO 710 as selective dopaminergic ligand for binding and autoradiographic studies.
DO 710, a benzamide derivative previously shown to display a dopamine antagonistic potency superior to that of sulpiride, was 3H-labeled. Its use as radioligand was assessed in membrane binding and autoradiographic studies. The compound displayed relatively high affinity (Kd 2 nM) and pronounced selectivity for dopamine receptors (distinct from D-1 receptors) as well as low non-specific binding particularly evidenced in autoradiographic experiments. Hence [3H](-)-DO 710 displays distinct technical advantages over commonly used dopaminergic radioligands.
Animals↗
3H-(-)DO 710 discriminates guanine nucleotide sensitive and insensitive dopamine binding sites.
(-)DO 710, a substituted benzamide derivative which discriminates dopamine D-2 and D-4 binding sites (Sokoloff et al. 1984), and antagonises in a differential manner several apomorphine-induced behavioral responses (Schwartz et al. 1984) was tritiated and used namely to differentially label the D-4 site. In striatum the 3H-(-)DO 710 saturation curve was best explained by the presence of two classes of sites with a 7-fold difference in affinity (Kd values of 3.0 nM and 0.42 nM) and Bmax values of 316 and 106 fmol X mg protein-1, respectively which correspond to the D-2 and D-4 sites (Sokoloff et al. 1984). In spite of its limited selectivity, 3H-(-)DO 710 in low concentration (0.2 nM) could be used to preferentially label striatal D-4 site as shown by the inhibition potencies of discriminant benzamide derivatives (DBD), significantly higher than at pituitary D-2 site (receptor) whereas classical neuroleptics including metoclopramide were equally potent at both sites. The affinity of a variety of agonists for striatal sites labeled with 0.2 nM 3H-(-)DO 710 generally differed from their affinity for the two states of the pituitary D-2 receptor (with high and low affinity for agonists, respectively); compounds like lisuride, N-propylnorapomorphine or pergolide had very high affinity for the striatal 3H-(-)DO 710 site. In pituitary from oestradiol-treated rats, where only D-2 site occurs, only the low-affinity site for 3H-(-)DO 710 (Kd = 2.8 nM) was found.(ABSTRACT TRUNCATED AT 250 WORDS)
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