The same mutation in Gsalpha and transducin alpha reveals behavioral differences between these highly homologous G protein alpha-subunits.

Mutating Arg-238 to Glu (R238E) in the switch 3 region of a transducin alpha (*Talpha) in which 27 aa of the GTPase domain have been replaced with those of the alpha-subunit of the inhibitory G protein 1 (Gi1alpha), was reported to create an alpha-subunit that is resistant to activation by GTPgammaS, is devoid of resident nucleotide, and has dominant negative (DN) properties. In an attempt to create a DN stimultory G protein alpha (Gsalpha) with a single mutation we created Gsalpha-R265E, equivalent to *Talpha-R238E. Gsalpha-R265E has facilitated activation by GTPgammaS, a slightly facilitated activation by GTP but much reduced receptor plus GTP stimulated activation, and an apparently unaltered ability to interact with receptor as seen in ligand binding studies. Further, the activity profile of Gsalpha-R265E is that of an alpha-subunit with unaltered or increased GTPase activity. The only change in Gsalpha that is similar to that in *Talpha is that the apparent affinity for guanine nucleotides is decreased in both proteins. The molecular basis of the changed properties are discussed based on the known crystal structure of Gsalpha and the changes introduced by the same mutation in a *Talpha (Gtalpha*) with only 23 aa from Gi1alpha. Gtalpha*-R238E, with four fewer mutations in switch 3, was reported to show no evidence of DN properties, is activated by GTPgammaS, and has reduced GTPase activity. The data highlight a critical role for the switch 3 region in setting overall properties of signal-transducing GTPases.

A Potent N-(piperidin-4-yl)-1H-pyrrole-2-carboxamide Inhibitor of Adenylyl Cyclase of G. lamblia: Biological Evaluation and Molecular Modelling Studies.

In this work, we report a derivative of N-(piperidin-4-yl)-1H-pyrrole-2-carboxamide as a new inhibitor for adenylyl cyclase of Giardia lamblia which was obtained from a study using structural data of the nucleotidyl cyclase 1 (gNC1) of this parasite. For such a study, we developed a model for this specific enzyme by using homology techniques, which is the first model reported for gNC1 of G. lamblia. Our studies show that the new inhibitor has a competitive mechanism of action against this enzyme. 2-Hydroxyestradiol was used as the reference compound for comparative studies. Results in this work are important from two points of view. on the one hand, an experimentally corroborated model for gNC1 of G. lamblia obtained by molecular modelling is presented; on the other hand, the new inhibitor obtained is an undoubtedly excellent starting structure for the development of new metabolic inhibitors for G. lamblia.

On the roles of Mg in the activation of G proteins.

In this review, we highlight the evolution of our knowledge about the way Mg(2+) participates in the activation of heterotrimeric G proteins, beginning with its requirement in hormonal stimulation of fat cell adenylyl cyclase (1969) and ending with knowledge that incorporates information obtained from site directed mutagenesis and examination of the crystal structures of G proteins (2010). Our current view is that, as it seeks to fill its octahedral coordination shell, Mg acts as a keystone locking the G protein-α subunits into a conformation in which Gα dissociates from the Gßγ dimer, is competent in regulating effectors, and acquires GTPase activity. The latter is the result of moving the backbone carbonyl group of the Mg-coordinating threonine into a location in space that positions the hydrolytic water so as to facilitate the water's nucleophilic attack that leads to hydrolysis of the link between the ß and γ phosphates of guanosine triphosphate (GTP). The role of the backbone carbonyl group of the Mg-coordinating threonine is equi-hierarchical with a similar and long-recognized role of the Switch II glutamine δ amide carbonyl group. Disruption of either leads to loss of GTPase activity.

Ganglioside glycosyltransferases and newly synthesized gangliosides are excluded from detergent-insoluble complexes of Golgi membranes.

GEM (glycosphingolipid-enriched microdomains) are specialized detergent-resistant domains of the plasma membrane in which some gangliosides concentrate. Although genesis of GEM is considered to occur in the Golgi complex, where the synthesis of gangliosides also occurs, the issue concerning the incorporation of ganglioside species into GEM is still poorly understood. In this work, using Chinese hamster ovary K1 cell clones with different glycolipid compositions, we compared the behaviour with cold Triton X-100 solubilization of plasma membrane ganglioside species with the same species newly synthesized in Golgi membranes. We also investigated whether three ganglioside glycosyltransferases (a sialyl-, a N-acetylgalactosaminyl- and a galactosyl-transferase) are included or excluded from GEM in Golgi membranes. Our data show that an important fraction of plasma membrane G(M3), and most G(D3) and G(T3), reside in GEM. Immunocytochemical examination of G(D3)-expressing cells showed G(D3) to be distributed as cold-detergent-resistant patches in the plasma membrane. These patches did not co-localize with a glycosylphosphatidylinositol-anchored protein used as GEM marker, indicating a heterogeneous composition of plasma membrane GEM. In Golgi membranes we were unable to find evidence for GEM localization of either ganglioside glycosyltransferases or newly synthesized gangliosides. Since the same ganglioside species appear in plasma membrane GEM, it was concluded that in vivo nascent G(D3), G(T3) and G(M3) segregate from their synthesizing transferases and then enter GEM. This latter event could have taken place shortly after synthesis in the Golgi cisternae, along the secretory pathway and/or at the cell surface.

Membrane distribution of epidermal growth factor receptors in cells expressing different gangliosides.

Gangliosides have been found to reside in glycosphingolipid-enriched microdomains (GEM) of the plasma membrane and to be involved in the regulation of epidermal growth factor receptor (EGFr or ErbB1) activity. To gain further insight into the mechanisms involved in EGFr modulation by gangliosides, we investigated the distribution of EGFr family members in the plasma membrane of CHO-K1 cells, which were genetically modified to express different ganglioside molecules or depleted of glycolipids. Our data demonstrate that at least four different sets of endogenously expressed gangliosides, including GD3, did not have a significant effect on EGFr distribution in the plasma membrane. In addition, using confocal microscopy analysis we clearly demonstrated that the EGFr co-localizes only to a minor extent with GD3. We also explored the endogenous expression, in wild-type CHO-K1 cells, of the orphan receptor ErbB2 (which is the preferred heteroassociation partner of all other ErbB proteins) and the effect of GD3 expression on its membrane distribution. Our results showed that CHO-K1 cells endogenously express ErbB2 and that expression of the GD3 affected, to some extent, the membrane distribution of endogenous ErbB2. Finally, our findings support the notion that most EGFr are excluded from GEM, while an important fraction of ErbB2 is found to be associated with these microdomains in membranes from CHO-K1 cells.

GD3 expression in CHO-K1 cells increases growth rate, induces morphological changes, and affects cell-substrate interactions.

We have generated a panel of CHO-K1 cell clones with different glycolipid compositions by stable transfection of appropriate glycosyltransferases and studied the morphological and growth phenotype of a clone stably expressing Sial-T2. Compared with the GM3 expressing parental cells, Sial-T2 transfectants show low expression of GM3 and neo expression of GD3 and GT3. These cells show about 60% reduction of the mean cell area, and about 2-fold increase of the mean colony area and growth rate. Cells over expressing Sial-T2 showed a flattened appearance, and with time in culture they detached from the substrate leaving adhered material that was GD3 immunoreactive. No apoptotic or proteome differences could be detected in the Sial-T2 transfectants. Thus, increased expression of GD3 and GT3 influence parameters of growth and social behavior of CHO-K1 cells. However, the molecular and cellular basis underlying these influences requires further investigation.