LABORATORIOS DE PRIMER NIVEL

Many extracellular signals affecting the cytoskeleton, including proteins, peptides and small molecules, bind to trans-membrane receptor molecules on the surface of the cell. A major receptor type is characterized by seven hydrophobic regions that potentially form membrane-spanning a helices. These receptors couple to effector molecules via heterotrimeric guanosine nucleotide-binding proteins (G-proteins).

Haploid cells of the yeast Saccharomyces cerevisiae respond to mating pheromones produced by other cells of the opposite mating type by arresting in the G1 phase of the cell cycle, altering surface protein distribution and forming a pear-shaped cell known as a 'shmoo'. The mating type of the cells is controlled by a single genetic locus with two alleles. Cells with the MATa allele secrete a-factor and respond to a-factor whilst cells with the MAT a allele secrete a-factor and respond to a-factor. The a-factor and a-factor are peptides of 12 and 13 amino acids respectively which bind to receptors encoded by STE2 (a-factor receptor) and STE3 (a-factor receptor) genes. The two receptors do not have significant sequence similarity but both appear to contain seven hydrophobic regions. In addition, genes encoding proteins with sequence similarity with heterotrimeric G-proteins, SCGl or GPAl (Ga), STE4 (GP) and STE18 (Gy) are also involved or are required for the mating response (118, 119).

The slime mold Dictyostelium discoideum has also been widely used to study signal transduction pathways controlling the cytoskeleton. When cells are stimulated by the chemoattractants cAMP or folate the actin cytoskeleton rearranges within seconds, prior to the polarisation of the cell and orientation in the chemoattractant gradients (120). Cloning and

sequence analysis oï di Dictyostelium cAMP receptor indicates the presence of seven putative transmembrane helices, similar to other vertebrate G-protein coupled receptors. The receptor appears to be essential for chemotaxis since strains transformed with anti-sense RNA for the receptor are unable to aggregate (1 2 1).

1.10 Heterotrimeric G-proteins.

Ligand binding to receptors of the seven transmembrane family initiates a complex series of events that are mediated by activation of heterotrimeric G proteins. G proteins consist of 3 subunits: a (which binds guanosine nucleotide), p and y. Interaction of the cytoplasmic domains of the receptor-ligand complex promotes dissociation of GTP/GDP from the a subunit and its replacement. At normal cytosolic concentrations, GTP is more likely to bind to the a subunit than is GDP. On binding GTP the a subunit dissociates from the Py subunits allowing both G„ and Gp.^ to regulate effector proteins such as phosphodiesterases, phospholipase Cp, adenylyl cyclases, phospholipase A2, kinases and ion channels. The a

subunit posses intrinsic GTPase activity and will hydrolyse the terminal phosphate of GTP to produce bound GDP and free phosphate. The a subunit bound to GDP has high affinity for the py complex which therefore re-associate, inhibiting subunit interactions with effectors and enhancing the complex binding to the receptor (1 2 2).

The specificity of the effector systems activated is determined by the types of a and py subunits of the G protein. There are at least 2 1 distinct a subunits encoded by 17 genes

that have been divided according to sequence similarity into 4 major groups G,, Gj, Gq and Gi2- In addition, there are at least five p and six y subunits, producing 30 possible Py

combinations, although not all p and y subunits interact so the number of different pairs is lower.

G proteins may be activated by more than one type of receptor, whilst conversely a single receptor may activate more than one G-protein type . Activation of effector systems by a subunits appears to be more specific. Reconstitution of Ga, into phospholipid vesicles with effector proteins show that it stimulates adenylyl cyclase and voltage-gated Ca^^ channels from skeletal muscle. Less is known about direct interactions of the family although they are thought to inhibit adenylyl cyclase, activate channels and inhibit Ca^^ channels, whilst transducins («j family members expressed in photoreceptors) activate cGMP-specific

phosphodiesterase. Members of the family activate phospholipase Cp when reconstituted in phospholipid vesicles, whilst « 1 2 regulates Na^/K^ exchange (123). In contrast, py dimers

activate effector systems, such as channels, adenylyl cyclase, phospholipase Cp, phospholipase Aj, phosphoinositide 3-kinase and p-adrenergic receptor kinase with little or no specificity in vitro (124).

Evidence suggesting heterotrimeric G proteins mediate cytoskeletal changes has come mostly from lower eukaryotes and from the use of cholera and pertussis toxins that ADP- ribosylate G proteins. Pheromone responses by the yeast Saccharomyces cerevisae are blocked by null mutations in STE4 and STE18 genes. Protein sequence analysis indicate similarities between the products of STE4 and STE18 and Gp and Gy respectively. Constitutive activation of the mating response by the Ga homologue indicates that the a subunit acts as a negative regulator of py dimer-mediated activation of signal transduction pathways (?). Heterotrimeric G-proteins are also essential for chemotaxis of Dictyostelium cells: fgdA mutants which lack G„2, a G protein implicated in phospholipase C activation, are

unable to aggregate in response to cyclic AMP (cAMP) stimulation (125). The non- hydrolysable GTP analogues, GTPyS and GppNHp, mimic some cAMP-induced effects, suggesting they are mediated by G proteins (126).