A limited spectrum of mutations causes constitutive activation of the yeast alpha-factor receptor.

Activation of G protein coupled receptors (GPCRs) by binding of ligand is the initial event in diverse cellular signaling pathways. To examine the frequency and diversity of mutations that cause constitutive activation of one particular GPCR, the yeast alpha-factor receptor, we screened libraries of random mutations for constitutive alleles. In initial screens for mutant receptor alleles that exhibit signaling in the absence of added ligand, 14 different point mutations were isolated. All of these 14 mutants could be further activated by alpha-factor. Ten of the mutants also acquired the ability to signal in response to binding of desTrp(1)¿Ala(3)alpha-factor, a peptide that acts as an antagonist toward normal alpha-factor receptors. Of these 10 mutants, at least eight alleles residing in the third, fifth, sixth, and seventh transmembrane segments exhibit bona fide constitutive signaling. The remaining alleles are hypersensitive to alpha-factor rather than constitutive. They can be activated by low concentrations of endogenous alpha-factor present in MATa cells. The strongest constitutively active receptor alleles were recovered multiple times from the mutational libraries, and extensive mutagenesis of certain regions of the alpha-factor receptor did not lead to recovery of any additional constitutive alleles. Thus, only a limited number of mutations is capable of causing constitutive activation of this receptor. Constitutive and hypersensitive signaling by the mutant receptors is partially suppressed by coexpression of normal receptors, consistent with preferential association of the G protein with unactivated receptors.

Aminoglycoside antibiotics bound to aminoglycoside-detoxifying enzymes and RNA adopt similar conformations.

Conformations of ribostamycin and isepamicin, aminoglycoside antibiotics, bound to an aminoglycoside antibiotic, 3'-phosphotransferase, were determined by transferred nuclear Overhauser effect spectroscopy and molecular modeling. Two major conformers of enzyme-bound ribostamycin, a neomycin-group aminoglyeoside were observed. The 3'- and 5"-OH groups (reactive hydroxyl groups) in the conformers are placed in approximate locations. One of the conformers is similar to the structure of paromomycin bound to a 27-nucleotide piece of ribosomal RNA that represents the A-site of the small ribosomal subunit, where rings A and C are in an orthogonal arrangement. Isepamicin, a kanamycin-group aminoglycoside antibiotic, also showed two major enzyme-bound conformations. Both conformations were similar to those observed for bound isepamicin in the active site of an aminoglycoside(6')-acetyl transferase-Ii. Conformations of other RNA-bound kanamycin-group aminoglycosides were also similar to the enzyme-bound conformations of isepamicin. These observations suggest that aminoglycosides may adopt similar conformations when bound to RNA and protein targets. This may have significant implications in the design of enzyme inhibitors and/or antibiotics.