Lakshmidevi Pulakat

Identification of an interaction between the angiotensin II receptor sub-type AT2 and the ErbB3 receptor, a member of the epidermal growth factor receptor family

To identify the proteins that interact and mediate angiotensin II receptor AT2-specific signaling, a random peptide library was screened by yeast-based Two-Hybrid protein-protein interaction assay technique. A peptide that shared significant homology with the amino acids located between the residues Gly-Xaa-Gly-Xaa-Xaa-Gly721 and Lys742, the residues predicted to be important for ATP binding of the ErbB3 and ErbB2 receptors, was identified to be interacting with the AT2 receptor. The interaction between the human ErbB3 receptor and the AT2 receptor was further confirmed using the cytoplasmic domain (amino acids 671-782) of the human ErbB3 receptor. Moreover, an AT2 receptor peptide that spans the amino acids 226-363, (spans the third ICL and carboxy terminal domain) could also interact with the AT2 receptor in a yeast Two-Hybrid protein-protein interaction assay. Studies using mutated and chimeric AT2 receptors showed that replacing the third intracellular loop (ICL) of the AT2 receptor with that of the AT1 abolishes the interaction between the ErbB3 and the AT2 in yeast Two-Hybrid protein-protein interaction assay. Thus the interaction between the AT2 receptor and the ErbB3 receptor seems to require the region spanning the third ICL and carboxy terminus of the AT2 receptor. Since the third ICL of the AT2 receptor is essential for exerting its inhibitory effects on cell growth, possible involvement of this region in the interaction with the cytoplasmic domain of the ErbB3 receptor suggests a novel signaling mechanism for the AT2 receptor mediated inhibition of cell growth. Furthermore, since both the AT2 and the ErbB3 receptors are expressed during fetal development, we propose that the existence of direct interaction between these two receptors may play a role in the regulation of growth during the initial stages of development.

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.

Role of C-terminal cytoplasmic domain of the AT2 receptor in ligand binding and signaling

A stop codon at position 322 was introduced to generate a truncated, C‐terminal‐deleted AT2 receptor. Expression studies in Xenopus oocytes showed that C‐terminal‐deleted AT2 had reduced affinity to [125I]angiotensin II (K d=1.7 nM) and enhanced binding of the AT2‐specific peptidic ligand [125I]CGP42112A (K d=0.097 nM). AT2 activation by angiotensin II resulted in reduction of cGMP levels in oocytes and this reduction was further enhanced by C‐terminal deletion, implying that the C‐terminus may have a negative effect on the AT2‐mediated cGMP reduction. Moreover, interaction of the AT2 with the ATP‐binding domain of the human ErbB3 receptor in yeast two‐hybrid assay was abolished by C‐terminal deletion. In summary, the C‐terminal cytoplasmic tail of AT2 modulates its ligand binding and signaling properties.

Role of the third intracellular loop of the angiotensin II receptor subtype AT2 in ligand-receptor interaction.

Angiotensin II (Ang II) receptor subtypes AT1 and AT2 share 34% overall homology, but the least homology is in their third intracellular loop (3rd ICL). In an attempt to elucidate the role of the 3rd ICL in determining the similarities and differences in the functions of the AT1 and the AT2 receptors, we generated a chimeric receptor in which the 3rd ICL of the AT2 receptor was replaced with that of the AT1 receptor. Ligand‐binding properties and signaling properties of this receptor were assayed by expressing this receptor in Xenopus oocytes. Ligand‐binding studies using [125I‐Sar1‐Ile8] Ang II, a peptidic ligand that binds both the AT1 and the AT2 receptor subtypes, and 125I‐CGP42112A, a peptidic ligand that is specific for the AT2 receptor, showed that the chimeric receptor has lost affinity to both ligands. However, IP3 levels of the oocytes expressing the chimeric receptor were comparable to the IP3 levels of the oocytes expressing the AT1 receptor, suggesting that the chimeric receptors could couple to phospholipase C pathway in response to Ang II. We have shown previously that the nature of the amino acid present in the position 215 located in the fifth transmembrane domain (TMD) of the AT2 receptor plays an important role in determining its affinity to different ligands. Our results from the ligand‐binding studies of the chimeric receptor further support the idea that the structural organization of the region spanning the 5th TMD and the 3rd ICL of the AT2 receptor has an important role in determining the ligand‐binding properties of this 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.

Isolation and characterization of nif DK::kanamycin and nitrogen fixation proficient Azotobacter vinelandii strain, and its implication on the status of multiple chromosomes in Azotobacter.

Several lines of experimental analyses on the ploidy status of Azotobacter vinelandii genome lead to the conclusion that it contains more than 40 copies of its chromosome and therefore it is a polyploid organism. The genetic evidence argues against the existence of polyploidy in these cells since the segregation pattern of genetic markers under lack of selection pressure mimic that of haploids. However, when A. vinelandii was made Nif− by inserting a kanamycin resistance marker gene in the nifDK sequence and the cells were selected for kanamycin resistance and Nif+ phenotype, we were able to score colonies that are both kanamycin resistant and Nif+. Therefore, when the cells were subjected to forced double selection of the same locus, they behaved as if they carried at least two chromosomes, one carrying the kanamycin resistance marker in the nifDK genes and the other carrying the intact nifDK genes. These analyses suggested that at least a diploidy status can be induced in these cells under selection pressure.

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.

Activation of vanadium nitrogenase expression in Azotobacter vinelandii DJ54 revertant in the presence of molybdenum

Azotobacter vinelandii carries three different and genetically distinct nitrogenase systems on its chromosome. Expression of all three nitrogenases is repressed by high concentrations of fixed nitrogen. Expression of individual nitrogenase systems is under the control of specific metal availability. We have isolated a novel type of A. vinelandii DJ54 revertant, designated A. vinelandii BG54, which carries a defined deletion in the nifH gene and is capable of diazotrophic growth in the presence of molybdenum. Inactivation of nifDK has no effect on growth of this mutant strain in nitrogen‐free medium suggesting that products of the nif system are not involved in supporting diazotrophic growth of A. vinelandii BG54. Similar to the wild type, A. vinelandii BG54 is also sensitive to 1 mM tungsten. Tn5‐B21 mutagenesis to inactivate the genes specific to individual systems revealed that the structural genes for vnf nitrogenase are required for diazotrophic growth of A. vinelandii BG54. Analysis of promoter activity of different nif systems revealed that the vnf promoter is activated in A. vinelandii BG54 in the presence of molybdenum. Based on these data we conclude that A. vinelandii BG54 strain utilizes vnf nitrogenase proteins to support its diazotrophic growth.