Colette Pantaloni

Neuroblastoma Differentiation Involves the Expression of Two Isoforms of the α‐Subunit of Go

The regulation of GTP‐binding proteins (G proteins) was examined during the course of differentiation of neuroblastoma N1E‐115 cells. N1E‐115 cell membranes possess three Bordetella pertussis toxin (PTX) substrates assigned to α‐subunits (Gα) of Go (a G protein of unknown function) and “Gi (a G protein inhibitory to adenylate cyclase)‐like” proteins and one substrate of Vibrio cholerae toxin corresponding to an α‐subunit of Gs (a G protein stimulatory to adenylate cyclase). In undifferentiated cells, only one form of Goα was found, having a pI of 5.8. Goα content increased by approximately twofold from the undifferentiated state to 96 h of cell differentiation. This is mainly due to the appearance of another Goα form having a pI of 5.55. Both Goα isoforms have similar sizes on sodium dodecyl sulfate‐polyacrylamide gels, are recognized by polyclonal antibodies to bovine brain Goα, are ADP‐ribosylated by PTX, and are covalently myristylated in whole N1E‐115 cells. In addition, immunofluorescent staining of N1E‐115 cells with Goα antibodies revealed that association of Goα with the plasma membrane appears to coincide with the expression of the most acidic isoform and morphological cell differentiation. In contrast, the levels of both Giα and Gsα did not significantly change, whereas that of the common β‐subunit increased by ∼ 30% over the same period. These results demonstrate specific regulation of the expression of Goα during neuronal differentiation.

Multiple species and isoforms of Bordetella pertussis toxin substrates

In purified G proteins from bovine brain cortex the ADP-ribosylated substrates of Bordetella pertussis toxin (PT) can be resolved in three polypeptides by polyacrylamide gel electrophoresis: a 39 kDa major substrate, corresponding to Go alpha and two others (40 and 41 kDa) assigned to alpha subunits of Gi-like proteins. These three polypeptides were also detected in membranes of normal cells or tissues from neuronal and endocrine origins. In contrast, in membranes from other origins, only two PT substrates at 41 and 40 kDa were resolved; the latter being the most abundant ADP-ribosylated substrate in human platelets and C6 glioma cells. In these cells, electrophoretic patterns of PT-radiolabeled proteolytic fragments derived from the 40 kDa peptide were different to those from the 39 and 41 kDa polypeptides of purified G proteins. However, isoelectrofocusing and two dimensional analyses showed that the 40 kDa and 39 kDa (but not the 41 kDa) PT substrate of purified G proteins exhibited similar isoforms.

Tissue expression and phylogenetic appearance of the β and γ subunits of GTP binding proteins

Guanyl nucleotide binding protein Adenylate cyclase Phototransduction Hormonal transduction

Functional domains of the Gs α subunit : role of the C-terminus in the receptor-dependent and receptor-independent activation

We have developed a rapid and simple model for studying functional domains of Gs alpha subunit, the GTP binding protein involved in adenylyl cyclase activation. Cyc- membranes prepared from a variant S49 cell line which does not express the alpha subunit of Gs are reconstituted by the in vitro translated Gs alpha subunit. Since the messenger RNA used for in vitro translation is generated from in vitro transcription of the cDNA encoding Gs alpha subunit, it is possible to introduce genetic modifications at the nucleotide level and analyze their consequences at the amino-acid level on the functional properties of the protein. We have constructed mutated alpha chains which correspond to various deletions of the carboxy-terminal region. Removal of the 9 carboxy-terminal residues uncoupled the alpha subunit from the receptor whereas deletion of the 26 carboxy-terminal residues blocked any activation induced either in a receptor-dependent or in a receptor-independent manner.

Adaptive Response of Beta-Adrenergic Sensitive Adenylate Cyclase System

In the beta adrenergic adenylate cyclase system, persistent stimulation of the cells by catecholamines or analogs results in a decline of the cyclic AMP response. This phenomenon generally referred to as desensi-tization has been classified in two types by Perkins and coworkers (1). In the first one called heterologous, incubation with one agonist leads to an attenuated response to different multiple hormones operating via distinct receptors. In the second one referred to as homologous or agonist-specific, only the subsequent response to the desensitizing hormone is diminished while the efficiency of other hormones is unimpaired. This type of desensi-tization, characterized by a decrease in adenylate cyclase stimulation by beta-adrenergic agonists, is associated in most cases with a loss or disappearance of beta-adrenergic receptors (for reviews see 2,3,4): this phenomenon is called down-regulation. The down-regulated receptors can be recycled to the cell surface or destroyed within the cell (2,3,4).