Jennifer N Heerding

Mutational analysis of the angiotensin II type 2 receptor: contribution of conserved extracellular amino acids

While much work has been done examining the ligand-binding characteristics of the AT1 receptor, very little attention has been focused on the AT2 receptor. Both receptors bind angiotensin II (AngII) with identical affinities, but share only 34% homology. Although it is tempting to assume that conserved residues between the two subtypes are responsible for the binding of AngII, there is little data to support this view. To determine the commonalities in ligand binding of the AT1 and AT2 receptors, we have chosen several conserved extracellular amino acids which have been shown to be important in AngII binding [1,2] to the AT1 receptor for mutational studies of the AT2 receptor. Specifically, we have mutated tyrosine108 in extracellular loop 1 (ECL1), arginine182 in ECL2, and aspartate297 in ECL3 of the AT2 receptor in order to determine their contribution to AngII binding. In the AT2 receptor, mutation of tyrosine108 to an alanine resulted in a receptor with wild-type binding for AngII, while mutation of either arginine182 or aspartate297 drastically impaired AngII binding ( > 100 nM). These results demonstrate both similarities as well as clear differences between receptor subtypes in the contributions to AngII binding of several conserved extracellular amino acid residues.

Role of the amino terminus in ligand binding for the angiotensin II type 2 receptor.

Key amino terminal residues in type 1 (AT1) angiotensin II (AngII) receptors are not conserved within type 2 (AT2) receptors. We therefore characterized amino terminal mutants that are transiently expressed in COS-3 membranes. AT2 amino terminal deletion drastically reduced affinity for AngII, suggesting its importance for this subtype. AT1-AT2 amino terminal exchanges retained wild type AngII affinities (Kd ranging from 3-5 nM), indicating compensation despite substantial sequence dissimilarities. Finally, binding of AT2 selective ligands (CGP42112A and PD123319) was not dependent on the amino terminus.

Mutation of a conserved fifth transmembrane domain lysine residue (Lys215) attenuates ligand binding in the angiotensin II type 2 receptor.

A fifth transmembrane domain lysine residue is conserved in both the type 1 (AT1) and type 2 (AT2) angiotensin II (AngII) receptors. This lysine (Lys199) is believed to play a critical role in peptide binding for the AT1 receptor. To evaluate its possible role in the AT2 receptor, the analogous AT2 residue (Lys199) was changed to glutamine. This mutation greatly reduced the affinity for both 125I-AngII and 125I-Sar1,Ile8-AngII and abolished binding to the non-peptide 125I-PD122979. These data indicate that despite a relatively low homology of 34%, some commonalities in the binding mechanism for AngII may exist between the two subtypes.

Mutational analysis of the angiotensin type 2 receptor : contribution of conserved amino acids in the region of the sixth transmembrane domain

Angiotensin II (AngII) mediates its physiological actions through two receptor subtypes: the Type 1 (AT1) and Type 2 (AT2) receptors. The subtypes have identical affinities for AngII, while sharing only 34% homology. Mutagenesis has focused mainly on the AT1 receptor, identifying residues important for AngII binding. In contrast, relatively little is known of the binding mechanism of the AT2 receptor. It has been hypothesized that residues that are conserved between the two subtypes that have been shown to be important in the AT1 receptor may also contribute to AngII binding in the AT2 receptor as well. To test this hypothesis, the role of two conserved residues in the sixth transmembrane domain of the AT2 receptor in ligand binding were investigated: tryptophan 269 and aspartate 279. In contrast to the AT1 receptor, mutation of Trp269 in the AT2 receptor to an alanine had no effect on AngII binding, while mutation of Asp279 to alanine similarly impaired AngII binding in both receptors. However, the more sterically conservative substitution of Asp279 to asparagine in the AT2 receptor showed near wild type affinity. Based on this finding, we mutated Asp263 in the AT1 receptor to asparagine. Subsequent studies indicated that this more conservative mutation had no effect on AngII binding to the AT1 receptor. Collectively, these results demonstrate that although there may be commonalities in ligand binding between the AT1 and AT2 AngII receptors, there are also clear differences.

Differential agonist modulation of the cloned opioid receptors reveals distinct cellular mechanisms of receptor regulation

Abstract Tolerance is a limitation to the clinical use of opioids. A cellular basis of tolerance may involve receptor downregulation/desensitization after exposure to agonists. We have investigated the effects of agonist pretreatments of cells expressing the cloned rat mu, and mouse delta and kappa receptors on both subsequent radioligand binding and coupling to adenylyl cyclase. Agonist pretreatment of cells expressing kappa and delta receptors diminished subsequent labelling of thoroughly washed membranes with agonist. Antagonist binding to the delta receptor was also diminished, but antagonist binding to the kappa receptor was unaffected. Neither agonist nor antagonist binding to the mu receptor was affected by agonist pretreatment. The loss of agonist binding to the delta and kappa receptors was paralleled by a loss of the ability of agonists to inhibit forskolin-stimulated cAMP accumulation. The mu receptor, in contrast, did not functionally desensitize. Desensitization of the kappa, but not delta, receptor could be blocked by cotransfection of the receptors with a dominant negative mutant of β-adrenergic receptor kinase. The mechanisms underlying the development of tolerance to opioid agents are likely multiple and divergent for the different receptor types.