Role of amino acid residue 90 in bioactivity and receptor binding capacity of tumor necrosis factor mutants

We have previously produced two bioactive lysine-deficient mutants of TNF-alpha (mutTNF-K90R,-K90P) and found that these mutants have bioactivity superior to wild-type TNF (wtTNF). Because these mutants contained same amino acid except for amino acid 90, it is unclear which amino acid residue is optimal for showing bioactivity. We speculated that this amino acid position was exchangeable, and this amino acid substitution enabled the creation of lysine-deficient mutants with enhanced bioactivity. Therefore, we produced mutTNF-K90R variants (mutTNF-R90X), in which R90 was replaced with other amino acids, to assay their bioactivities and investigated the importance of amino acid position 90. As a result, mutTNF-R90X that replaced R90 with lysine, arginine and proline were bioactive, while other mutants were not bioactive. Moreover, these three mutants showed bioactivity as good as or better than wtTNF. R90 replaced with lysine or arginine had especially superior binding affinities. These results suggest that the amino acid position 90 in TNF-alpha is important for TNF-alpha bioactivity and could be altered to improve its bioactivity to generate a "super-agonist".     

Studies on the conformation of secretin: The position of the helical stretch

An analog of the C-terminal tricosapeptide of secretin, with aspartic acid replacing glutamic acid in position 9 and lysine substituted for arginine in position 21, was prepared. The synthesis was carried out in solution by stepwise chain lengthening with the application of the in situ technique. The ord-cd spectra of this new analog closely resemble the spectra of the tricosapeptide with the unaltered secretin sequence and of the analog in which only arginine-21 was replaced by lysine and of secretin itself. The incorporation of aspartic acid instead of glutamic acid-9 resulted in an N-terminal sequence that has a consïderably reduced probability of assuming a helical conformation. The observation that the helix content remained unchanged adds support to a model of secretin in which the helical stretch is near the C-terminus. The role of an acidic residue in position 9 is also discussed.     

Phosphorylation by guanosine 3':5'-monophosphate-dependent protein kinase of synthetic peptide analogs of a site phosphorylated in histone H2B

Analogs of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B were used to assess the substrate specificity of cGMP-dependent protein kinase. cGMP-dependent kinase phosphorylated the oligopeptide Arg-Lys-Arg-Ser32-Arg-Lys-Glu with favorable kinetic parameters as compared to those for cAMP-dependent kinase (Glass, D. B., and Krebs, E. G. (1979) J. Biol. Chem. 254, 9728-9738). The contribution of each amino acid to the ability of the peptide to be phosphorylated by cGMP-dependent or cAMP-dependent kinase was studied by replacement of individual residues and evaluation of the kinetic constants of the substituted peptides. Peptides containing acetylated lysine residues or nitroarginine residues were poor substrates for both kinases. Substitution of either arginine 29 or lysine 30 with alanine increased the Km values and decreased the Vmax values for both kinases. Substitution of lysine 34 with alanine increased the Vmax values for both kinases but did not affect the Km values for either enzyme. Substitution of the phosphorylatable serine with a threonine residue greatly depressed the Vmax for both kinases. Peptides in which arginine 31 or arginine 33 were replaced by an alanine residue revealed several apparent differences in the specificity requirements between cGMP-dependent and cAMP-dependent kinases.     

Solubility of hemoglobin S by sedminetation, equilibrium, and antisickling compounds.

The effect of the compounds guanidine, arginine, lysine, and aspartic acid and the salt arginine aspartate on the solubility of deoxyhemoglobin S (Hb S) was studied by sedimentation equilibrium at 20–22 °C. Guanidine and arginine were found to be most effective, whereas aspartic acid and lysine had only a small effect. The effectiveness of these compounds in solubilizing Hb S is relatively pH independent. It is unlikely that the small effect of lysine and aspartic acid on the solubility of Hb S can account for the antisickling properties of lysine and aspartic acid previously reported (Sophianopoulos, A. J., et al. (1974) Clin. Biochem.7, 112–118). The effect of guanidine and arginine is large enough to account for a large part of such antisickling properties (Sophianopoulos et al. (1974). The nonideality of concentrated hemoglobin solutions (up to 0.3 g cm^?3) has been studied in detail. By using the liganded as well as the unliganded forms of both Hb S and Hb A, it was found that the magnitude of the virial (nonideality) coefficients can change with varying solution conditions. A comparison of pure Hb S with hemolysates is made using viscosity and sedimentation velocity.     

Kinetics of chemical modification of arginine and lysine residues in calf thymus histone H1

The arginine and lysine residues of calf thymus histone H1 were modified with large molar excesses of 2,3-butanedione and O-methylisourea, respectively. Kinetic study of the modification reaction of the arginine residue revealed that the reaction is divided into the two pseudo-first-order processes. About a third (1 Arg) of the total arginine residues of the H1 molecule was rapidly modified without causing any detectable structural change of the molecule, and the slow modification of the remaining arginine residues (2 Arg) led to a loss of the folded structure of H1. In the case of lysine residue modification, 93% (56 Lys) of the total lysine residues of the H1 was modified with the same rate constant, while 7% (4 Lys) of lysine residue remained unmodified. When the reaction was performed in the presence of 6M guanidine-HCl, all of lysine residues were modified. It is concluded that the 2 arginine and 4 lysine residues resistant to modification are buried in interior regions of the H1 molecule and play an important role in the formation of the H1 globular structure, while the other 1 arginine and 56 lysine residues are exposed to solvent.     

Stimulation of Lysine Decarboxylase Production in Escherichia coli by Amino Acids and Peptides

A commercial hydrolysate of casein stimulated production of lysine decarboxylase (EC 4.1.1.18) by Escherichia coli B. Cellulose and gel chromatography of this hydrolysate yielded peptides which were variably effective in this stimulation. Replacement of individual, stimulatory peptides by equivalent amino acids duplicated the enzyme levels attained with those peptides. There was no indication of specific stimulation by any peptide. The peptides were probably taken up by the oligopeptide transport system of E. coli and hydrolyzed intracellularly by peptidases to their constituent amino acids for use in enzyme synthesis. Single omission of amino acids from mixtures was used to screen them for their relative lysine decarboxylase stimulating abilities. Over 100 different mixtures were evaluated in establishing the total amino acid requirements for maximal synthesis of lysine decarboxylase by E. coli B. A mixture containing all of the common amino acids except glutamic acid, aspartic acid, and alanine increased lysine decarboxylase threefold over an equivalent weight of casein hydrolysate. The nine most stimulatory amino acids were methionine, arginine, cystine, leucine, isoleucine, glutamine, threonine, tyrosine, and asparagine. Methionine and arginine quantitatively were the most important. A mixture of these nine was 87% as effective as the complete mixture. Several amino acids were inhibitory at moderate concentrations, and alanine (2.53 mM) was the most effective. Added pyridoxine increased lysine decarboxylase activity 30%, whereas other B vitamins and cyclic adenosine 5′-monophosphate had no effect.     

Purification of Synechocystis sp. strain PCC6308 cyanophycin synthetase and its characterization with respect to substrate and primer specificity

Synechocystis sp. strain PCC6308 cyanophycin synthetase was purified 72-fold in three steps by anion exchange chromatography on Q Sepharose, affinity chromatography on the triazine dye matrix Procion Blue HE-RD Sepharose, and gel filtration on Superdex 200 HR from recombinant cells of Escherichia coli. The native enzyme, which catalyzed the incorporation of arginine and aspartic acid into cyanophycin, has an apparent molecular mass of 240 +/- 30 kDa and consists of identical subunits of 85 +/- 5 kDa. The K(m) values for arginine (49 microM), aspartic acid (0.45 mM), and ATP (0.20 mM) indicated that the enzyme had a high affinity towards these substrates. During in vitro cyanophycin synthesis, 1.3 +/- 0.1 mol of ATP per mol of incorporated amino acid was converted to ADP. The optima for the enzyme-catalyzed reactions were pH 8.2 and 50 degrees C, respectively. Arginine methyl ester (99.5 and 97% inhibition), argininamide (99 and 96%), S-(2-aminoethyl) cysteine (43 and 42%), beta-hydroxy aspartic acid (35 and 37%), aspartic acid beta-methyl ester (38 and 40%), norvaline (0 and 3%), citrulline (9 and 7%), and asparagine (2 and 0%) exhibited an almost equal inhibitory effect on the incorporation of both arginine and aspartic acid, respectively, when these compounds were added to the complete reaction mixture. In contrast, the incorporation of arginine was diminished to a greater extent than that of aspartic acid, respectively, with canavanine (82 and 53%), lysine (36 and 19%), agmatine (33 and 25%), D-aspartic acid (37 and 30%), L-glutamic acid (13 and 5%), and ornithine (23 and 11%). On the other hand, canavanine (45% of maximum activity) and lysine (13%) stimulated the incorporation of aspartic acid, whereas aspartic acid beta-methyl ester (53%) and asparagine (9%) stimulated the incorporation of arginine. [(3)H]lysine (15% of maximum activity) and [(3)H]canavanine (13%) were incorporated into the polymer, when they were either used instead of arginine or added to the complete reaction mixture, whereas L-glutamic acid was not incorporated. No effect on arginine incorporation was obtained by the addition of other amino acids (i.e., alanine, histidine, leucine, proline, tryptophan, and glycine). Various samples of chemically synthesized poly-alpha,beta-D,L-aspartic acid served as primers for in vitro synthesis of cyanophycin, whereas poly-alpha-L-aspartic acid was almost inactive.     

Aspartic Acid 397 in Subunit B of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae Forms Part of a Sodium-binding Site,Is Involved in Cation Selectivity,and Affects Cation-binding Site Cooperativity

The Na⁺-pumping NADH:quinone complex is found in Vibrio cholerae and other marine and pathogenic bacteria. NADH:ubiquinone oxidoreductase oxidizes NADH and reduces ubiquinone, using the free energy released by this reaction to pump sodium ions across the cell membrane. In a previous report, a conserved aspartic acid residue in the NqrB subunit at position 397, located in the cytosolic face of this protein, was proposed to be involved in the capture of sodium. Here, we studied the role of this residue through the characterization of mutant enzymes in which this aspartic acid was substituted by other residues that change charge and size, such as arginine, serine, lysine, glutamic acid, and cysteine. Our results indicate that NqrB-Asp-397 forms part of one of the at least two sodium-binding sites and that both size and charge at this position are critical for the function of the enzyme. Moreover, we demonstrate that this residue is involved in cation selectivity, has a critical role in the communication between sodium-binding sites, by promoting cooperativity, and controls the electron transfer step involved in sodium uptake (2Fe-2S → FMN_C).     

Regulation of lysine transport by feedback inhibition in Saccharomyces cerevisiae.

A steady-state level of about 240 nmol/mg (dry wt) occurs during lysine transport in Saccharomyces cerevisiae. No subsequent efflux of the accumulated amino acid was detected. Two transport systems mediate lysine transport, a high-affinity, lysine-specific system and an arginine-lysine system for which lysine exhibits a lower affinity. Preloading with lysine, arginine, glutamic acid, or aspartic acid inhibited lysine transport activity; preloading with glutamine, glycine, methionine, phenylalanine, or valine had little effect; however, preloading with histidine stimulated lysine transport activity. These preloading effects correlated with fluctuations in the intracellular lysine and/or arginine pool: lysine transport activity was inhibited when increases in the lysine and/or arginine pool occurred and was stimulated when decreases in the lysine and/or arginine pool occurred. After addition of lysine to a growing culture, lysine transport activity was inhibited more than threefold in one-third of the doubling time of the culture. These results indicate that the lysine-specific and arginine-lysine transport systems are regulated by feedback inhibition that may be mediated by intracellular lysine and arginine.     

Relationship between amino acid production and phosphate-dissolving capacity of bacteria

The ability of soil bacteria to produce amino acids (alanine, aspartic acid, leucine, arginine, glutamic acid, and lysine) was related to the ability to dissolve inorganic phosphate. With the exception of lysine, amino acid production increased with increasing ability to dissolve phosphate.     

Cation permeability and cation-anion interactions in a mutant GABA-gated chloride channel from Drosophila.

To investigate the structural basis of anion selectivity of Drosophila GABA-gated Cl(-) channels, the permeation properties of wild-type and mutant channels were studied in Xenopus oocytes. This work focused on asparagine 319, which by homology is one amino acid away from a putative extracellular ring of charge that regulates cation permeation in nicotinic receptors. Mutation of this residue to aspartate reduced channel conductance, and mutation to lysine or arginine increased channel conductance. These results are consistent with an electrostatic interaction between this site and permeating anions. The lysine mutant, but not the arginine mutant, formed a channel that is permeable to cations, and this cannot be explained in terms of electrostatics. The lysine mutant had a 25-mV reversal potential in solutions with symmetrical Cl(-) and asymmetrical cations. The permeability ratio of K(+) to Cl(-) was determined as 0. 33 from reversal potential measurements in KCl gradients. Experiments with large organic cations and anions showed that cation permeation can only be seen in the presence of Cl(-), but Cl(-) permeation can be seen in the absence of permeant cations. Measurements of permeability ratios of organic anions indicated that the lysine mutant has an increased pore size. The cation permeability of the lysine-containing mutant channel cannot be accounted for by a simple electrostatic interaction with permeating ions. It is likely that lysine substitution causes a structural change that extends beyond this one residue to influence the positions of other channel-forming residues. Thus protein conformation plays an important role in enabling ion channels to distinguish between anions and cations.     

Importance of the lysine cluster in the translocation of anions through the pyrophosphate specific channel OprO

Pseudomonas aeruginosa is a Gram-negative bacterium with an intrinsic resistance towards antibiotics due to the lack of a large diffusion pores. Exchange of substances with the environment is done mainly through a set of narrow and substrate-specific porins in its outer membrane that filter molecules according to their size and chemical composition. Among these proteins are OprP and OprO involved in the selective uptake of mono- and pyrophosphates, respectively. Both proteins are homotrimers and each monomer features an hourglass-shaped channel structure including a periplasmic cavity with a lysine cluster. In this study, we focus on the characterization of this lysine cluster in OprO. The importance of these lysine residues was shown with alanine substitutions in single channel conductance experiments, by titration of mono- and pyrophosphate in multi-channel analysis and by molecular dynamics simulations. All obtained data demonstrated that the closer the mutated lysine residues are to arginine 133, the lower gets the single channel conductance. It was found that the ion flow through each monomer can follow two different lysine paths indicating that phosphate ions have a larger freedom on the periplasmic side of the constriction region. Our results emphasize the important role of the lysine residue 121 in the binding site together with arginine 133 and aspartic acid 94. An improved understanding of the ion mobility across these channels can potentially lead to an optimized permeation of (phosphonic acid containing) antibiotics through the outer membrane of P. aeruginosa and the development of new drug molecules.     

Mutational analysis of the cleavage sequence of the human immunodeficiency virus type 1 envelope glycoprotein precursor gp160.

The envelope glycoproteins of the human immunodeficiency virus (HIV) type 1 are synthesized as a precursor molecule, gp160, which is cleaved to generate the two mature envelope glycoproteins, gp120 and gp41. The cleavage reaction, which is mediated by a host protease, occurs at a sequence highly conserved in retroviral envelope glycoprotein precursors. We have investigated the sequence requirements for this cleavage reaction by introducing four single-amino-acid changes into the glutamic acid-lysine-arginine sequence immediately amino terminal to the site of cleavage. We have also examined the effects of these mutations on the syncytium formation induced by HIV envelope glycoproteins. Our results indicate that a glutamic acid to glycine change at gp120 amino acid 516, a lysine to isoleucine change at amino acid 517, and an arginine to lysine change at amino acid 518 affect neither gp160 cleavage nor syncytium formation. The results obtained with the arginine to lysine change at amino acid 518 differ significantly from the results obtained with the same mutation at the envelope precursor cleavage site of a murine leukemia virus (E. O. Freed, and R. Risser, J. Virol. 61:2852-2856, 1987). An arginine to threonine mutation at gp120 amino acid 518, the terminal residue of gp120, abolishes both gp160 cleavage and syncytium formation. These findings demonstrate that despite its highly conserved nature, the basic pair of amino acids at the site of gp160 cleavage is not absolutely required for proper envelope glycoprotein processing. This report also supports the idea that cleavage of gp160 is required for activation of the HIV envelope fusion function.     

Biosynthesis of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin): mechanism of the cyanophycin synthetase reaction studied with synthetic primers.

Biosynthesis of the cyanobacterial nitrogen reserve cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is catalysed by cyanophycin synthetase, an enzyme that consists of a single kind of polypeptide. Efficient synthesis of the polymer requires ATP, the constituent amino acids aspartic acid and arginine, and a primer like cyanophycin. Using synthetic peptide primers, the course of the biosynthetic reaction was studied. The following results were obtained: (a) sequence analysis suggests that cyanophycin synthetase has two ATP-binding sites and hence probably two active sites; (b) the enzyme catalyses the formation of cyanophycin-like polymers of 25-30 kDa apparent molecular mass in vitro; (c) primers are elongated at their C-terminus; (d) the constituent amino acids are incorporated stepwise, in the order aspartic acid followed by arginine, into the growing polymer. A mechanism for the cyanophycin synthetase reaction is proposed; (e) the specificity of the enzyme for its amino-acid substrates was also studied. Glutamic acid cannot replace aspartic acid as the acidic amino acid, whereas lysine can replace arginine but is incorporated into cyanophycin at a much lower rate.     

Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase

The specificities of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase were probed using synthetic peptides corresponding to the sequence around phosphorylation sites 1 and 2 on pyruvate dehydrogenase [Tyr-His-Gly-His-Ser(P1)-Met-Ser-Asp-Pro-Gly-Val-Ser(P2)-Tyr-Arg]. The dephosphotetradecapeptide containing aspartic acid at position 8 was a better substrate for the kinase than was the tetradecapeptide containing asparagine at position 8. The apparent Km and V values for the two peptides were 0.43 and 6.1 mM and 2.7 and 2.4 nmol of 32P incorporated/min/mg, respectively. Methylation of the aspartic acid residue also increased the apparent Km of the tetradecapeptide about 14-fold. These results indicate that an acidic residue on the carboxyl-terminal side of phosphorylation site 1 is an important specificity determinant for the kinase. Phosphate was incorporated only into site 1 of the synthetic peptide by the kinase. The phosphatase exhibited an apparent Km of 0.28 mM and a V of 2.3 mumol of 32P released/min/mg for the phosphorylated tetradecapeptide containing aspartic acid. Methylation of the aspartic acid residue had no significant effect on dephosphorylation. The octapeptide and phosphooctapeptide produced by cleavage of the aspartyl-prolyl bond by formic acid were poorer substrates for the kinase and phosphatase than were the tetradecapeptide and phosphotetradecapeptide, respectively. Modification of the amino terminal by acetylation or lysine addition had only a slight effect on the kinase and phosphatase activities.     

Chemical modification and amino terminal sequence of calotropin DI from Calotropis gigantea

The effect of chemical modification of histidine, lysine, arginine, tryptophan and methionine residues on the enzymatic activity of calotropin DI has been studied. 1,3-Dibromoacetone inhibited the enzyme completely, indicating that a single histidine residue and a cysteine residue are involved in its catalytic activity. Its second bistidine residue was modified with diethyl pyrocarbonate without loss of activity. Modification of seven of its 13 lysine residues with 2,4,6-trinitrobenzene sulphonic acid led to 90% loss of its activity, but no single lysine residue appears to be essential for its activity. Four of the 12 arginine residues by 1,2-cyclohexanedione can be modified with little loss of activity. Modification of a single tryptophan residue and two methionine residues did not inhibit enzymatic activity. The blocked amino-terminal amino acid residue of calotropin DI has been identified as pyroglutamic acid. Its amino-terminal amino acid sequence to residue 14 has been determined and compared with that of papain. They show an extensive homology in their amino-terminal amino acid sequences.     

Dynamics of the amino acid composition of the medium during cultivation of isolated organs by the directed perfusion method]

The dynamics of the amino acid composition of the medium under conditions of adequate perfusion of the isolated organs of a dog (sternum, kidney and liver) was studied. It was found that after a 6-hour perfusion of the complex of organs the amount in the perfusion medium of such amino acids as histidine, lysine, alanine, considerably increased, whereas the amount of arginine, serine, aspartic acid, threonine with glutamine, isoleucine, proline, leucine and valine decreased as compared with their initial concentration. The dynamics of the amino acid medium composition during a 4-hour perfusion was studied in experiments with the isolated sternum. The concentration of alanine, lysine and histidine increased in the medium. At the same time there was seen a decrease in the concentration of serine, aspartic acid, isoleucine, tyrosine and phenyl-alanine.     

Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors.

The transmembrane PTPase HPTP beta differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354-amino acid, 41-kDa human PTP beta catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTP beta shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9-11 residues from lck, src, and PLC gamma with Km values of 2, 4, and 1 microM, some 40-200-fold lower than the other two PTPases. With kcat values of 30-205 s-1, the catalytic efficiency, kcat/Km, of the HPTP beta 41-kDa catalytic domain is very high, up to 5.7 x 10(7) M-1 s-1. The peptides corresponding to PLC gamma (766-776) and EGFR (1,167-1,177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTP beta catalytic domain. While exchange of the alanine residue at the +2 position of the PLC gamma (Km of 1 microM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35-fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 microM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the -2 position of the EGFR peptide decreased the Km value 34-fold to 3 microM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.     

Critical molecular regions for elicitation of the sweetness of the sweet-tasting protein, thaumatin I

Thaumatin is an intensely sweet-tasting protein. To identify the critical amino acid residue(s) responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using Pichia pastoris, in which alanine residues were substituted for lysine or arginine residues, and the sweetness of each mutant protein was evaluated by sensory analysis in humans. Four lysine residues (K49, K67, K106 and K163) and three arginine residues (R76, R79 and R82) played significant roles in thaumatin sweetness. Of these residues, K67 and R82 were particularly important for eliciting the sweetness. We also prepared two further mutant thaumatin I proteins: one in which an arginine residue was substituted for a lysine residue, R82K, and one in which a lysine residue was substituted for an arginine residue, K67R. The threshold value for sweetness was higher for R82K than for thaumatin I, indicating that not only the positive charge but also the structure of the side chain of the arginine residue at position 82 influences the sweetness of thaumatin, whereas only the positive charge of the K67 side chain affects sweetness.     

Replacement of lysine-181 by aspartic acid in the third transmembrane region of endothelin type B receptor reduces its affinity to endothelin peptides and sarafotoxin 6c without affecting G protein coupling.

A conserved aspartic acid residue in the third transmembrane region of many of the G protein-coupled receptors has been shown to play a role in ligand binding. In the case of endothelin receptors, however, a lysine residue replaces this conserved aspartic acid residue. To access the importance of this residue in ligand binding, we have replaced it with an aspartic acid in the rat endothelin type B (ETb) receptor by PCR mediated mutagenesis. The binding characteristics and functional properties of both the wild type and mutant receptors were determined in COS-7 cells transiently expressing the cloned receptor cDNAs. Using 125I-ET-1 as the radioactive peptide ligand in displacement binding studies, the wild type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three endothelin peptides (ET-1, ET-2, and ET-3) and sarafotoxin 6c (SRTX 6c). Interestingly, the mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM) but displayed a much larger increase in IC50 value for SRTX 6c (> 10 uM). The lysine mutant receptor still elicited full inositol phosphate (IP) turnover responses in the presence of saturating concentrations of endothelins (10 nM of ET-1, 100 nM of ET-2, or 1 uM of ET-3), indicating that the mutation (K181D) did not affect the coupling of mutant receptor to the appropriate G protein. These results demonstrate that lysine-181 on the receptor is important for binding ET peptides; however, it is required for binding the ETb selective agonist-SRTX 6c.