br Agonist binding ETA activation
Agonist binding ETA activation is promoted by binding of the endogenous peptidergic agonists to their orthosteric binding site on the receptor. ET1 and ET2 (Trp6-Leu7-ET1) bind with equal high affinity to ETA, whereas the third endogenous isopeptide ET3 (Thr2-Phe4-Thr5-Tyr6-Lys7-Tyr14-ET1) binds with considerably less affinity 3, 5. This agonist–receptor binding is not influenced by GTP, which reduces the affinity of many GPCRs for their agonists. Truncation, amidation or extension of the C-terminal Trp21 of ET1 abolishes binding. The linear analogue 4Ala-ET1 (in which both disulfide bonds are absent due to replacement of all four cysteine residues by alanine) does not bind to ETA. These observations indicate that the C-terminal tail, selected NCT-503 in the N-terminal loop and both disulfide bonds are all required for polyvalent binding of endogenous peptides to ETA16, 25, 26. The orthosteric binding site of ETA would thus contain more than one functional domain. Studies using site-directed mutagenesis, chimeric receptors and photoaffinity labeling indicate that these orthosteric binding domains are located between TM helices 1, 2, 3 and 7, and between TMs 4, 5 and 6 of ETA, respectively 25, 27, 28, 29, 30. Hilal-Dandan et al. proposed that the ultimate polyvalent ET1–ETA complex is preceded by one or more transitory conformations . However, the equilibrium dissociation constants and the structure–affinity relationships of these intermediates have not been defined. Results of binding studies using intact radioactively labeled ET1 provide information about the kinetics of the formation of the ultimate complex and include the influences of receptor activation on agonist affinity 32, 33. The rate of association between ET1 and ETA is comparable to that for angiotensin II (another potent vasoactive peptide) acting on the AT1a GPCR. The rate of dissociation (0.0005min–1) is, however, 100 times slower. As a result, complexes composed of radioactively labeled ET1 and ETA cannot be dissociated by cold agonists, and their half-life ranges from 7 to 77h . ET1 thus binds tightly to ETA.
Model of receptor binding and activation It has been proposed that ETAs display an inactive conformation (R) or an active conformation (R*) that can bind one or more G proteins (G) and other effector proteins (Figure 1a). An orthosteric agonist (e.g. ET1) binds sequentially and polyvalently to R and promotes its activation (R→R* conversion). A first transitory complex (R-et) is a prerequisite for subsequent formation of the quasi-irreversible R-ET complex (Figure 1b). Although R-et binding and G-R*-et function might exhibit typical class A GPCR behavior, R-ET binding and G-R*-ET function do not. The initial transitory complexes might exhibit dynamic equilibrium and susceptibility to desensitization and tolerance. By contrast, the ultimate complexes are tight and their effects are long-lasting and persistent (Figure 1b). Figure 1c,d illustrates the effects of a neutral competitive antagonist and an allosteric modulator in this situation, respectively. Recent work by our research team revealed functional observations  that, in combination with earlier structure–affinity relationships 25, 34, suggest roles for different parts of the agonist molecule and different domains of ETA in (i) the dynamic interaction, (ii) ultimate tight binding and (iii) activation of the receptor. We therefore propose that ET1 consists of two functional parts (Figure 2). One part of the agonist would be responsible for association with ETA. This would concentrate another part of the agonist at another domain of the receptor, which could then bind tightly and activate the receptor. For various other endogenous peptides that act on class B GPCRs, the reported structure–activity relationships are in line with the presence of distinct ‘address’ and ‘message’ domains 35, 36, 37, 38. For ET1, such detailed information is not available yet, but structural analyses indicate a flexibility of the agonist molecule  that might be essential for the two parts of the agonist to interact sequentially with ETA (Figure 2). In the event that ET1 consists of functional parts separated by a hinge region , the orthosteric binding site of ETA contains at least two functional domains (Figure 2). This suggestion is supported by studies using engineered receptors and photoaffinity labeling 27, 28, 29, 30, 34.