Current model of IR activation and proposed binding mechanism for S961. A

<p>. Current model of IR activation. The four blue circles represent the receptor binding sites (sites 1 and 2) seen from a top view. Insulin is depicted as a yellow circle. For a detailed explanation of binding sites 1 and 2, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051972#pone.0051972-Whitesell1" target="_blank">[24]</a>. <b>B</b>. Proposed binding mechanism for S961. The four blue circles represent the receptor binding sites (sites 1 and 2) seen from a top view. For a detailed explanation of binding sites 1 and 2, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051972#pone.0051972-Whitesell1" target="_blank">[24]</a>. The S961 peptide (Site 1–2 peptide) is shown as two connected yellow circles. At concentrations of 1–10 nM, S961 crosslinks the receptor, leading to agonist activity. At concentrations of above 10 nM, the higher flexibility of S961 in comparison to the insulin molecule allows simultaneous crosslinking of both pairs of binding sites, leading to an inactive conformation and antagonism. The corresponding activation and inactivation sigmoids are also shown. <b>C</b>. Orientation of peptide binding sites. If site 1 is located N-terminally and site 2 C-terminally, a longer distance between the binding sites in S961 in comparison to S661 can be achieved.</p

Agonistic (mitogenic) effect of S961 in MCF-7 cells.

<p>Cells were stimulated with increasing concentrations of S961 or IGF-I. The graph is representative for three experiments. The increased response for S961 at 1 nM compared to the response at the three lowest concentrations is statistically significant (P<0.001, two-tailed t-test). Data points represent means of triplicate determinations. Error bars show one standard deviation.</p

S961 stimulates IR and Akt phosphorylation in CHO-hIR cells.

<p>Cells were stimulated with increasing concentrations of HI or S961. <b>A</b>-<b>E</b>, IR tyrosine phosphorylation. The 6 tyrosine phosphorylation sites which were examined were Y972 in the juxtamembrane domain, Y1158 and Y1162/1163 in the tyrosine kinase domain, and Y1328 and Y1334 in the C-terminal tail end of the IR. <b>F</b>, Akt phosphorylation. Phosphorylation of Ser437 is known to be required for Akt activation. Panels <b>A</b>-<b>E</b>: the increased tyrosine phosphorylation of the IR was significant (compared to 0.0001 nM, 0.001 nM and 0.01 nM S961, P<0.05*, P<0.01**, P<0.001***, two-tailed t-test). Panel <b>F</b>: the increased serine phosphorylation of Akt was significant (compared to 0.0001 nM, 0.001 nM and 0.01 nM S961, P<0.01**, two-tailed t-test). Data points represent average of three independent experiments, each comprising triplicate determinations. Error bars show one standard deviation.</p

S961 has antagonist as well as agonist activity on IR-mediated mitogenic effect in L6-hIR cells. A,

<p>"10 nM S961" and "100 nM S961" curves: Cells were pretreated for 2h with 10 nM or 100 nM S961, and stimulated with increasing concentrations of insulin (as indicated on the x-axis) in the continued presence of S961. "HI" curve, insulin stimulation only (without S961). "DMSO" curve, insulin stimulation with equal volume DMSO added instead of S961. <b>B,</b> "10 nM S961" and "100 nM S961" curves: Cells were pretreated for 2h with 10 nM or 100 nM S961, and stimulated with increasing concentrations of IGF-I (as indicated on the x-axis) in the continued presence of S961. "IGF-I" curve, IGF-I stimulation only (without S961). "DMSO" curve, IGF-I stimulation with equal volume DMSO added instead of S961. <b>C,</b> "0.01 nM HI", "0.025 nM HI" and "0.05 nM HI" curves: Cells were pretreated for 2 h with increasing concentrations of S961 (as indicated on the x-axis), and stimulated with 0.01 nM, 0.025 nM or 0.05 nM HI in the continued presence of S961. "S961 alone" curve, insulin was omitted. <b>D</b>, "1 nM HI", "10 nM HI" and "100 nM HI" curves: Cells were pretreated for 2 h with increasing concentrations of S961 (as indicated on the x-axis), and stimulated with 1 nM, 10 nM or 100 nM HI in the continued presence of S961. "S961 alone" curve, insulin was omitted. <b>A</b> and <b>B,</b> Graphs are representative for three independent experiments, each experiment comprising triplicate determinations of each ligand concentration. <b>C,</b> The graph is performed in triplicates once. <b>D,</b> The graph is representative for two independent experiments each performed in triplicates. Error bars indicate one standard deviation.</p

S961 did not stimulate lipogenesis in rat adiopocytes.

<p>Primary rat adipocytes were stimulated with increasing concentrations of S961or S661 alone or in combination with 1 nM insulin. Insulin alone was included as a reference. The graph is representative of two independent experiments each comprising duplicate determinations. Error bars show one standard deviation.</p

S961 did not stimulate glycogen synthesis in differentiated adipocytes or in muscle cells. A

<p>, Differentiated 3T3-L1 adipocytes were stimulated with increasing concentrations of HI, IGF-I or S961. The graph is representative of two independent experiments each comprising duplicate determinations. Error bars show one standard deviation. <b>B</b>, L6-hIR muscle cells were stimulated with increasing concentrations of S961/S661 alone or in combination with 3 nM insulin. The graph is representative of two independent experiments each comprising triplicate determinations. Error bars show one standard deviation.</p

Structural Basis of the Aberrant Receptor Binding Properties of Hagfish and Lamprey Insulins

The insulin from the Atlantic hagfish (<i>Myxine glutinosa</i>) has been one of the most studied insulins from both a structural and a biological viewpoint; however, some aspects of its biology remain controversial, and there has been no satisfying structural explanation for its low biological potency. We have re-examined the receptor binding kinetics, as well as the metabolic and mitogenic properties, of this phylogenetically ancient insulin, as well as that from another extant representative of the ancient chordates, the river lamprey (<i>Lampetra fluviatilis</i>). Both insulins share unusual binding kinetics and biological properties with insulin analogues that have single mutations at residues that contribute to the hexamerization surface. We propose and demonstrate by reciprocal amino acid substitutions between hagfish and human insulins that the reduced biological activity of hagfish insulin results from unfavorable substitutions, namely, A10 (Ile to Arg), B4 (Glu to Gly), B13 (Glu to Asn), and B21 (Glu to Val). We likewise suggest that the altered biological activity of lamprey insulin may reflect substitutions at A10 (Ile to Lys), B4 (Glu to Thr), and B17 (Leu to Val). The substitution of Asp at residue B10 in hagfish insulin and of His at residue A8 in both hagfish and lamprey insulins may help compensate for unfavorable changes in other regions of the molecules. The data support the concept that the set of unusual properties of insulins bearing certain mutations in the hexamerization surface may reflect those of the insulins evolutionarily closer to the ancestral insulin gene product