Mutations of the DRY motif that preserve beta 2-adrenoceptor coupling.

The largest family of G protein coupled receptors is characterized by the presence of the DRY motif at the boundary between the third transmembrane region and the second intracellular loop. Inspection of a recent alignment containing 620 receptors reveals that arginine is the best conserved amino acid of this trio. Disease causing receptor mutations have been identified in which this arginine had been replaced by another amino acid. Those receptors were unable to stimulate G protein activity suggesting an obligatory role of this amino acid in the process. One such mutation was identified in the V2 vasopressin receptor (V2R). The R137H V2R binds vasopressin with wild type-like affinity but fails to stimulate Gs. Because interaction with G proteins have been found to modulate agonist binding affinity of adrenergic receptors, arginine 131 was replaced by histidine in the human beta 2-adrenergic receptor to explore the hypothesis that this mutation may dissociate the G protein effect on binding from G protein activation. Surprisingly, this substitution increased agonist binding affinity preserving wild type like coupling of the receptor. Arginine could also be substituted by other amino acids without loss of coupling to Gs demonstrating an unexpected lack of selectivity in the human beta 2-adrenergic receptor protein, and ruling out a requirement for the side chain of arginine in signalling to G proteins.

Palmitoylation of the V2 vasopressin receptor.

Palmitoylation of the V2 vasopressin receptor (V2R) and its functional role were investigated in transfected cells. Palmitoylation was assessed by incubating transfected cells with [3H]palmitic acid and immunoprecipitating the receptor with an antibody raised against a portion of the third intracellular loop of V2R. Wild-type and nonglycosylated V2R yielded tritium signals at 45-55 and 40 kDa, respectively, demonstrating that the V2R is palmitoylated and that receptor palmitoylation is independent of glycosylation. Substitution of CC341/342 for serines eliminated receptor palmitoylation, whereas replacement of a single amino acid, C341S or C342S, restored partial palmitoylation. Saturation binding assays revealed decreased cell surface expression of the nonpalmitoylated receptor compared with the wild-type; this effect was more pronounced when a truncated form of V2R (G345ter) was studied. The presence of either cysteine residue (C341S or C342S) elevated receptor expression to normal levels, most likely due to the partial restoration of palmitoylation. Ligand binding affinity, hormone-induced stimulation of adenylyl cyclase activity, receptor internalization, and desensitization were not affected by the absence of palmitoylation. No increase but rather a slight decrease in the extent of receptor palmitoylation was detected after exposure to vasopressin. It was concluded that the V2R is palmitoylated in both cysteines, each cysteine is palmitoylated independently from the other, and palmitoylation enhances cell surface expression of the V2R.

Diverse effects of mutation on the activity of the Escherichia coli export chaperone SecB.

The Escherichia coli SecB protein binds newly synthesized precursor maltose-binding protein (preMBP) and promotes its rapid export from the cytoplasm. Site-directed mutagenesis of two regions of SecB was carried out to better understand factors governing the SecB.preMBP interaction. 30 aminoacyl substitution mutants were analyzed, revealing two distinct classes of secB mutants. Substitutions at the alternating positions Phe-74, Cys-76, Val-78, or Gln-80 reduced the ability of SecB to form stable complexes with preMBP, but caused only mild defects in the rate of MBP export from living cells. The pattern revealed by this class of mutants suggests that a primary binding site for preMBP is hydrophobic and contains beta-sheet secondary structure. In contrast, substitutions at Asp-20, Glu-24, Leu-75, or Glu-77 caused a severe slowing in the rate of MBP export but did not disrupt SecB.preMBP complex formation. These largely acidic residues may function to regulate the opening of a preprotein binding site, allowing both high affinity preprotein binding and rapid dissociation of SecB.preprotein complexes at the membrane translocation site.