Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation.

We have suggested previously that both the negatively and positively charged residues of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif play an important role in the activation process of the alpha(1b)-adreneric receptor (AR). In this study, R143 of the E/DRY sequence in the alpha(1b)-AR was mutated into several amino acids (Lys, His, Glu, Asp, Ala, Asn, and Ile). The charge-conserving mutation of R143 into lysine not only preserved the maximal agonist-induced response of the alpha(1b)-AR, but it also conferred high degree of constitutive activity to the receptor. Both basal and agonist-induced phosphorylation levels were significantly increased for the R143K mutant compared with those of the wild-type receptor. Other substitutions of R143 resulted in receptor mutants with either a small increase in constitutive activity (R143H and R143D), impairment (R143H, R143D), or complete loss of receptor-mediated response (R143E, R143A, R143N, R143I). The R413E mutant displayed a small, but significant increase in basal phosphorylation despite being severely impaired in receptor-mediated response. Interestingly, all the arginine mutants displayed increased affinity for agonist binding compared with the wild-type alpha(1b)-AR. A correlation was found between the extent of the affinity shift and the intrinsic activity of the agonists. The analysis of the receptor mutants using the allosteric ternary complex model in conjunction with the results of molecular dynamics simulations on the receptor models support the hypothesis that mutations of R143 can drive the isomerization of the alpha(1b)-AR into different states, highlighting the crucial role of this residue in the activation process of the receptor.

The caveolin-binding motif of the pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is required for in vivo export of cholesteryl acetate.

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in different physiological processes. Their molecular mode of action, however, is poorly understood. Saccharomyces cerevisiae expresses three members of this superfamily, pathogen-related yeast (Pry)1, -2, and -3. We have recently shown that Pry function is required for the secretion of cholesteryl acetate and that Pry proteins bind cholesterol and cholesteryl acetate, suggesting that CAP superfamily members may generally act to bind sterols or related small hydrophobic compounds. Here, we analyzed the mode of sterol binding by Pry1. Computational modeling indicates that ligand binding could occur through displacement of a relatively poorly conserved flexible loop, which in some CAP family members displays homology to the caveolin-binding motif. Point mutations within this motif abrogated export of cholesteryl acetate but did not affect binding of cholesterol. Mutations of residues located outside the caveolin-binding motif, or mutations in highly conserved putative catalytic residues had no effect on export of cholesteryl acetate or on lipid binding. These results indicate that the caveolin-binding motif of Pry1, and possibly of other CAP family members, is crucial for selective lipid binding and that lipid binding may occur through displacement of the loop containing this motif.

Mode of interaction of the Gαo subunit of heterotrimeric G proteins with the GoLoco1 motif of Drosophila Pins is determined by guanine nucleotides.

Drosophila GoLoco motif-containing protein Pins is unusual in its highly efficient interaction with both GDP- and the GTP-loaded forms of the α-subunit of the heterotrimeric Go protein. We analysed the interactions of Gαo in its two nucleotide forms with GoLoco1-the first of the three GoLoco domains of Pins-and the possible structures of the resulting complexes, through combination of conventional fluorescence and FRET measurements as well as through molecular modelling. Our data suggest that the orientation of the GoLoco1 motif on Gαo significantly differs between the two nucleotide states of the latter. In other words, a rotation of the GoLoco1 peptide in respect with Gαo must accompany the nucleotide exchange in Gαo. The sterical hindrance requiring such a rotation probably contributes to the guanine nucleotide exchange inhibitor activity of GoLoco1 and Pins as a whole. Our data have important implications for the mechanisms of Pins regulation in the process of asymmetric cell divisions.

Club copy of letter of condolence to C. Morgan Arnold

A copy of a letter sent to a Mr. [C. Morgan] Arnold from the St. Catharines Garrison Club offering condolences on the death of his son, Major Henry M. Arnold, at the battle of Paardeberg, South Africa.