Mechanism Of Enhanced Bioactivity Of Tsh Analogs

A long-standing postulate held that charge-charge interactions are of major importance in the interaction between TSH and TSH receptor (Rees Smith et al. 1988). Similar

Figure 3. Schematic of a range of dose-response curves to two agonists (Kenakin 2003). Various degree of difference in potency and efficacy (Vmax) is dependent on assays sensitivity determined by coupling efficiency and receptor number. In poorly coupled systems (assay a), the lower-efficacy agonist (dotted line) shows almost no response. In intermediate-sensitivity assays (b-d), both are partial agonists, whereas in high-sensitivity assays (f and g) both are full agonists.

Figure 3. Schematic of a range of dose-response curves to two agonists (Kenakin 2003). Various degree of difference in potency and efficacy (Vmax) is dependent on assays sensitivity determined by coupling efficiency and receptor number. In poorly coupled systems (assay a), the lower-efficacy agonist (dotted line) shows almost no response. In intermediate-sensitivity assays (b-d), both are partial agonists, whereas in high-sensitivity assays (f and g) both are full agonists.

interactions between additional basic residues in TSH superactive analogs and specific acidic residues in the receptor, yet to be fully characterized, are likely involved in prolonged dissociation rates and enhanced receptor activation and signaling.

Largely unpublished studies indicated that new TSH superactive analogs are receptor specific and inactive at related LH and FSH receptors; analog concentrations up to 1000 fold higher than activating TSH receptors did not result in detectable stimulation. TSH analogs showed their enhanced activity at the TSH receptor from different species (i.e. human, rat, mouse), suggesting that various changes described during mammalian evolution of TSH receptor (Kaczur et al. 2003) are probably not involved in the phenomenon of analog "superactivity." In addition, an enhanced in vitro activity of TSH analogs was observed in normal media and in buffers with various salt concentrations. The effect of substitutions on the TSH in vitro bioactivity was in most cases highly correlated with their effect on receptor binding affinity. TSH superactive analogs are also more potent than standard rec-hTSH stimulators of inositol phosphate pathway. The difference in bioactivity between TSH analogs and wile-type hormone was demonstrated using cells with TSH receptor and largely depleted pool of the negatively charged cell surface proteoglycans. This further indicated that the mechanism of enhanced activity is not dependent on their interaction with heparan sulfate proteoglycans recognizing various motifs of basic residues (Cardin and Weintraub 1989; Bozon et al. 1998).

Although the exact mechanism of interaction of TSH analogs with TSH receptor has not yet been fully clarified specific interaction modes of TSH analogs with TSH receptor have been discovered. Our initial model of TSH-TSH receptor interaction was generally supported by "charge-reversal mutagenesis" and other approaches (Grossmann et al. 1997; Costagliola et al. 2002; Smits et al. 2003)) (Figure 4).

Figure 4. Schematic configuration of TSH-TSHR complex. Two parallel P-hairpin loops ofthe a-subunit (aLl, aL3) are located in the lower part of the model and may participate in the interaction with C-terminal portion of extracellular domain and the extracellular loops ofthe receptor transmembrane domain. Two loops of the are shown in the upper part with proposed binding site within the concave of leucine rich-repeats modeled by Kajava et al (Kajava et al. 1995).

Figure 4. Schematic configuration of TSH-TSHR complex. Two parallel P-hairpin loops ofthe a-subunit (aLl, aL3) are located in the lower part of the model and may participate in the interaction with C-terminal portion of extracellular domain and the extracellular loops ofthe receptor transmembrane domain. Two loops of the are shown in the upper part with proposed binding site within the concave of leucine rich-repeats modeled by Kajava et al (Kajava et al. 1995).

Analogous amino acid substitutions in the a-subunit were also performed in hCG, LH and FSH. Although significant increases in respective gonadotropin affinities for their cognate receptors were observed, in contrast to TSH analogs, differences were smaller for both single and combined substitutions (Szkudlinski et al. 1996). Consequently the effects of single mutations were more difficult to detect and only selected combinations were later confirmed at various laboratories (Heikoop et al. 1999).

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