Using green fluorescent proteins to study G-protein-coupled receptor localization and trafficking.

G-protein-coupled receptors (GPCRs) mediate a diverse array of biological functions as a result of their ability to respond selectively to extracellular stimuli, which ultimately results in cell-specific activation of signaling cascades. Generally, GPCR activation is followed rapidly by a loss of responsiveness, termed desensitization, which is then followed by a period of recovery or resensitization. These changes in signaling potential are tightly regulated, primarily via mechanisms that involve GPCR phosphorylation and trafficking to distinct locations within the cell. Tagging of GPCRs with the green fluorescent protein (GFP) has enabled the direct visualization of real-time trafficking of GPCRs in living cells. Such analyses have provided crucial insight into the mechanisms involved in controlling GPCR function.

The GTP hydrolysis defect of the Saccharomyces cerevisiae mutant G-protein Gpa1(G50V).

The Saccharomyces cerevisiae haploid cell response to pheromone involves two seven-transmembrane-domain pheromone receptors that couple to a heterotrimeric G protein. The G50V mutation in the G protein alpha subunit (G(alpha)), Gpa1p, is analogous to the p21(ras) transforming mutation Gly-->Val 12, and has been extensively examined for the phenotypes it produces in yeast cells. Here we have characterized the Gpa1(G50V) mutant protein in vitro by examining GTPgammaS binding, GDP exchange, GTP occupancy and guanosine triphosphatase (GTPase) activity. Compared to wild-type (WT) Gpa1p, Gpa1(G50V)p was found to have a moderately reduced GTPase activity and increased GTP occupancy, while GTPgammaS binding and GDP exchange were not significantly altered. The yeast regulator of G protein Signalling (RGS) protein, Sst2p, was also expressed and purified, and found to have a significantly reduced ability to stimulate the initial rate of GTP hydrolysis of Gpa1(G50V)p compared to its effect on WT Gpa1p. Probing conformational transitions by a protease sensitivity assay suggested that Gpa1(G50V)p did not bind the transition state mimetic GDP/AlF(4)(-) as efficiently as the WT Gpa1p. These biochemical results can explain many of the known gpa1(G50V) yeast cell phenotypes.

Role of clathrin-mediated endocytosis in agonist-induced down-regulation of the beta2-adrenergic receptor.

Previous studies have demonstrated that non-visual arrestins function as adaptors in clathrin-mediated endocytosis to promote agonist-induced internalization of the beta2-adrenergic receptor (beta2AR). Here, we characterized the effects of arrestins and other modulators of clathrin-mediated endocytosis on down-regulation of the beta2AR. In COS-1 and HeLa cells, non-visual arrestins promote agonist-induced internalization and down-regulation of the beta2AR, whereas dynamin-K44A, a dominant-negative mutant of dynamin that inhibits clathrin-mediated endocytosis, attenuates beta2AR internalization and down-regulation. In HEK293 cells, dynamin-K44A profoundly inhibits agonist-induced internalization and down-regulation of the beta2AR, suggesting that receptor internalization is critical for down-regulation in these cells. Moreover, a dominant-negative mutant of beta-arrestin, beta-arrestin-(319-418), also inhibits both agonist-induced receptor internalization and down-regulation. Immunofluorescence microscopy analysis reveals that the beta2AR is trafficked to lysosomes in HEK293 cells, where presumably degradation of the receptor occurs. These studies demonstrate that down-regulation of the beta2AR is in part due to trafficking of the beta2AR via the clathrin-coated pit endosomal pathway to lysosomes.

Visualization of agonist-induced sequestration and down-regulation of a green fluorescent protein-tagged beta2-adrenergic receptor.

To date, the visualization of beta2-adrenergic receptor (beta2AR) trafficking has been largely limited to immunocytochemical analyses of acute internalization events of epitope-tagged receptors in various transfection systems. The development of a beta2AR conjugated with green fluorescent protein (beta2AR-GFP) provides the opportunity for a more extensive optical analysis of beta2AR sequestration, down-regulation, and recycling in cells. Here we demonstrate that stable expression of beta2AR-GFP in HeLa cells enables a detailed temporal and spatial analysis of these events. Time-dependent colocalization of beta2AR-GFP with rhodamine-labeled transferrin and rhodamine-labeled dextran following agonist exposure demonstrates receptor distribution to early endosomes (sequestration) and lysosomes (down-regulation), respectively. The observed temporal distribution of beta2AR-GFP was consistent with measures of receptor sequestration and down-regulation generated by radioligand-receptor binding assays. Cells stimulated with different beta-agonists revealed time courses of beta2AR-GFP redistribution reflective of the intrinsic activity of each agonist.

Analysis of the receptor binding domain of Gpa1p, the G(alpha) subunit involved in the yeast pheromone response pathway.

The Saccharomyces cerevisiae G protein alpha subunit Gpa1p is involved in the response of both MATa and MAT alpha cells to pheromone. We mutagenized the GPA1 C terminus to characterize the receptor-interacting domain and to investigate the specificity of the interactions with the a- and alpha-factor receptors. The results are discussed with respect to a structural model of the Gpa1p C terminus that was based on the crystal structure of bovine transducin. Some mutants showed phenotypes different than the pheromone response and mating defects expected for mutations that affect receptor interactions, and therefore the mutations may affect other aspects of Gpa1p function. Most of the mutations that resulted in pheromone response and mating defects had similar effects in MATa and MAT alpha cells, suggesting that they affect the interactions with both receptors. Overexpression of the pheromone receptors increased the mating of some of the mutants tested but not the wild-type strain, consistent with defects in mutant Gpa1p-receptor interactions. The regions identified by the mating-defective mutants correlated well with the regions of mammalian G(alpha) subunits implicated in receptor interactions. The strongest mating type-specific effects were seen for mutations to proline and a mutation of a glycine residue predicted to form a C-terminal beta turn. The analogous beta turn in mammalian G(alpha) subunits undergoes a conformational change upon receptor interaction. We propose that the conformation of this region of Gpa1p differs during the interactions with the a- and alpha-factor receptors and that these mating type-specific mutations preclude the orientation necessary for interaction with one of the two receptors.

Genetic relationships between the G protein beta gamma complex, Ste5p, Ste20p and Cdc42p: investigation of effector roles in the yeast pheromone response pathway.

The Saccharomyces cerevisiae G protein beta gamma dimer, Ste4p/Ste18p, acts downstream of the alpha subunit, Gpa1p, to activate the pheromone response pathway and therefore must interact with a downstream effector. Synthetic sterile mutants that exacerbate the phenotype of ste4-ts mutations were isolated to identify proteins that functionally interact with Ste4p. The identification of a ste18 mutant indicated that this screen could identify proteins that interact directly with Ste4p. The other mutations were in STE5 and the STE20 kinase gene, which act near Ste4p in the pathway, and a new gene called STE21. ste20 null mutants showed residual mating, suggesting that another kinase may provide some function. Overexpression of Ste5p under galactose control activated the pheromone response pathway. This activation was dependent on Ste4p and Ste18p and partially dependent on Ste20p. These results cannot be explained by the linear pathway of Ste4p-->Ste20p-->Ste5p. Overexpression of Cdc42p resulted in a slight increase in pheromone induction of a reporter gene, and overexpression of activated forms of Cdc42p resulted in a further twofold increase. Mutations in pheromone response pathway components did not suppress the lethality associated with the activated CDC42 mutations, suggesting that this effect is independent of the pheromone response pathway.

Functional analysis of the sporulation-specific SPR6 gene of Saccharomyces cerevisiae.

The SPR6 gene of Saccharomyces cerevisiae encodes a moderately abundant RNA that is present at high levels only during sporulation. The gene contains a long open reading frame that could encode a hydrophilic protein approximately 21 kDa in size. This protein is probably produced by the yeast, because the lacZ gene of Escherichia coli is expressed during sporulation when fused to SPR6 in the expected reading frame. SPR6 is inessential for sporulation; mutants that lack SPR6 activity sporulate normally and produce viable ascospores. Nonetheless, the SPR6 gene encodes a function that is relevant to sporulating cells; the wild-type allele can enhance sporulation in strains that are defective for several SPR functions. SPR6 is located on chromosome V, 14.4 centimorgans centromere-distal to MET6.