Narasaiah Gavini

Role of Lys215 located in the fifth transmembrane domain of the AT2 receptor in ligand–receptor interaction

Studies on ligand-receptor interaction of Angiotensin II (Ang II) receptor type 1 have shown that for peptidic ligands to bind this receptor they must interact via their C-terminal carboxylate group to the positively charged side chain of the Lysine residue 199 located in the fifth transmembrane domain of this receptor. In the Ang II receptor type AT2, this Lysine residue is conserved at position 215 in the fifth transmembrane domain. To determine the specific mechanism of ligand binding to the Angiotensin II receptor type AT2, mutated AT2 receptors were generated in which the Lys215 was replaced with glutamic acid, glutamine, alanine and arginine. The ability of these mutated receptors to bind peptidic ligands 125I-[Sar1-Ile8]Ang II (non-specific for AT2 receptor type), 125I-CGP42112A (AT2 receptor specific) and the non-peptidic ligand PD123319 (AT2 receptor specific) was evaluated by expressing these receptors in Xenopus oocytes and performing binding assays. The Lys215Glu and Lys215Gln mutants of AT2 receptor lost their affinity to 125I-[Sar1-Ile8]Ang II, but retained their affinity to 125I-CGP42112A and PD123319. In contrast, Lys215Arg mutant retained its affinity to 125I-[Sar1-Ile8]Ang II, but exhibited lower affinity to 125I-CGP42112A. The Lys215Ala mutant lost its affinity to both 125I-[Sar1-Ile8]Ang II and 125I-CGP42112A. These results suggest that the binding mechanism of 125I-[Sar1-Ile8]Ang II to AT2 receptor is similar to that of AT1 receptor since an amino acid with positively charged side chain (Lys or Arg) located in the fifth transmembrane domain is required for this ligand to bind AT2 receptor. In contrast, although CGP42112A is a peptidic ligand, it does not require an interaction between its C-terminal carboxylate group and the positively charged side-chain of an amino acid in the fifth transmembrane domain for its binding to AT2 receptor.

Identification of the region of AT2 receptor needed for inhibition of the AT1 receptor-mediated inositol 1,4,5-triphosphate generation

Increase in the intracellular inositol triphosphate (IP3) levels in Xenopus oocytes in response to expression and activation of rat angiotensin II (Ang II) receptor AT1 was inhibited by co‐expression of rat AT2 receptor. To identify which region of the AT2 was involved in this inhibition, ability of three AT2 mutants to abolish this inhibition was analyzed. Deletion of the C‐terminus of the AT2 did not abolish this inhibition. Replacing Ile249 in the third intracellular loop (3rd ICL) of the AT2 with proline, corresponding amino acid in the AT1, in the mutant M6, resulted in slightly reduced affinity to [125I]Ang II (K d=0.259 nM), however, did not abolish the inhibition. In contrast, replacing eight more amino acids in the 3rd ICL of the AT2 (at positions 241–244, 250–251 and 255–256) with that of the AT1 in the mutant M8, not only increased the affinity of the AT2 receptor to [125I]Ang II (K d=0.038 nM) but also abolished AT2‐mediated inhibition. Interestingly, activation of the M8 by Ang II binding also resulted in increase in the intracellular IP3 levels in oocytes. These results imply that the region of the 3rd ICL of AT2 spanning amino acids 241–256 is sufficient for the AT2‐mediated inhibition of AT1‐stimulated IP3 generation. Moreover, these nine mutations are also sufficient to render the AT2 with the ability to activate phospholipase C.

Role of Asp297 of the AT2 receptor in high-affinity binding to different peptide ligands.

To determine how ligand-receptor interaction is affected by the charges of the amino acids at position 2 of the ligands and position 297 of the AT2 receptor, we generated the Asp297Lys mutant of AT2 and a ligand SarAsp(2)Ile. Asp297Lys mutant lost affinity to Ang II and SarIle however retained partial affinity to 125I-CGP42112A. The SarAsp(2)Ile had high affinity to Asp297Lys (IC(50)3.5nM) and partial affinity to the AT2 (IC(50)15nM). Therefore, not only the charge, but also the length of the side arms of the amino acids at position 2 of the ligand and position 297 of the receptor affect their interaction.

Identification of a second site compensatory mutation in the Fe-protein that allows diazotrophic growth of Azotobacter vinelandii UW97.

Azotobacter vinelandii UW97 is defective in nitrogen fixation due to a replacement of serine at position 44 by phenylalanine in the Fe‐protein [Pulakat, L., Hausman, B.S., Lei, S. and Gavini, N. (1996) J. Biol. Chem. 271, 1884–1889]. Serine residue 44 is located in a conserved domain that links the nucleotide binding site and the MoFe‐protein docking surface of the Fe‐protein. Therefore, it is possible that the loss of function by A. vinelandii UW97‐Fe‐protein may be caused by global conformational disruption or disruption of the conformational change upon MgATP binding. To determine whether it is possible to generate a functional nitrogenase complex via a compensating second site mutation(s) in the Fe‐protein, we have attempted to isolate genetic revertants of A. vinelandii UW97 that can grow on nitrogen‐free medium. One such revertant, designated A vinelandii BG9, encoded a Fe‐protein that retained the Ser44Phe mutation and also had a second mutation that caused the replacement of a lysine at position 170 by glutamic acid. Lysine 170 is highly conserved and is located in a conserved region of the Fe‐protein. This region is implicated in stabilizing the MgATP‐induced conformation of the Fe‐protein and in docking to the MoFe‐protein. Further complementation analysis showed that the Fe‐protein mutant that retained serine 44 but contained the substitution of lysine at position 170 by glutamic acid was also non‐functional. Thus, neither Ser44Phe nor Lys170Glu mutants of Fe‐protein were functional; however, the Fe‐protein in A. vinelandii BG9 that contained both substitutions could support diazotrophic growth on the strain.

Role of ribosome release in the basal level of expression of the Escherichia coli gene pheA

In Escherichia coli, the expression of the phenylalanine biosynthetic operon pheA is regulated by an attenuation mechanism. In the presence of excess phenylalanine, gene expression was decreased to 10% of the fully deattenuated level. To understand the factors that determine the basal level of pheA expression, we examined the role of ribosome release from the leader peptide stop codon UGA. The transcriptional readthrough from the pheA attenuator decreased by over 2-fold in the presence of the defective release factor 2. However, a release factor 1 (UAG and UAA specific) mutation did not influence the basal level of pheA expression. These results support the proposal that the release of translating ribosomes from the leader peptide stop codon in stem 2 of the pheA attenuator plays a crucial role in determining the basal level of expression of this gene.