Multiple receptor states related to truncation covalent modification and mutation

In addition to the ligand-mediated changes in receptor conformation, processes like covalent modification, truncation and mutation are also prone to affect the way in which receptors interact with other molecules.

As an example of covalent modification, the phosphorylated receptor clearly represents another 'state' to the 'activated' receptor. Accordingly, 'activated' and

Figure 207 Left: Dissociation of [3H]candesartan from wild-type AT1 receptors (negative charge, polar Lys199) or mutated receptors (neutral, polar Gln199 or neutral non-polar Ala199) (Vauquelin et aL, 2002, reproduced by permission of Jraas Ltd.). Right: Lys199 to Gln mutation: drop in affinity of AT1 receptor antagonists versus their dissociation half-life for the wild-type receptor.

Figure 207 Left: Dissociation of [3H]candesartan from wild-type AT1 receptors (negative charge, polar Lys199) or mutated receptors (neutral, polar Gln199 or neutral non-polar Ala199) (Vauquelin et aL, 2002, reproduced by permission of Jraas Ltd.). Right: Lys199 to Gln mutation: drop in affinity of AT1 receptor antagonists versus their dissociation half-life for the wild-type receptor.

'phosphorylated' receptors are able to interact with distinct proteins and this provides a molecular basis to explain switches in cellular signalling during the sustained stimulation of a receptor. Whereas the 'activated' receptor prefers one or more specific G proteins, the phosphorylated receptor may:

• Trigger signal transduction pathways by interacting with non-G proteins (e.g. by forming P-arrestin-receptor complexes).

• Switch the signal between distinct G proteins. In this respect, work with the P2-adrenergic receptor indicates that the selectivity of receptor-G protein coupling

Figure 208 Graphical representation of the proposed interaction of Arg167 with the tetrazole moiety and of Lys199 with the carboxyl group of candesartan (Vauquelin et aL, 2002, reproduced by permission of Jraas Ltd.).

Figure 209 Possible mechanism underlying the 'switch' of the functional coupling of a given receptor with distinct G proteins. Reprinted from Pharmacology and Therapeutics, 99, Hermans, E., Biochemical and pharmacological control of the multiplicity of coupling at G protein-coupled receptors, 25-44. © (2003), with permission from Elsevier.

Figure 209 Possible mechanism underlying the 'switch' of the functional coupling of a given receptor with distinct G proteins. Reprinted from Pharmacology and Therapeutics, 99, Hermans, E., Biochemical and pharmacological control of the multiplicity of coupling at G protein-coupled receptors, 25-44. © (2003), with permission from Elsevier.

may be regulated by receptor phosphorylation (Figure 209). Whereas the PKA-phosphorylated receptor shows reduced ability to couple to Gs, it gains the ability to interact with Gj. It is likely that this switch mechanism serves to attenuate the initial Gs-mediated increase in cAMP accumulation by a Gi-dependent feedback inhibition. The receptor-Gi protein interaction will also initiate the activation of MAPK (via Py-mediated activation of a Src family tyrosine kinase).

In fact, any structural modification of a GPCR may affect its interaction with other molecules and it is not always easy to know whether the affected amino acids directly participated in the interaction or whether their modification produced a conformational change of the receptor. Examples of such altered interactions are widespread:

Truncation of the AT1a carboxyl terminus produces a receptor mutant that couples well to G1 and signals in response to Ang II, but exhibits vastly reduced internalization.

Deletion of part of TM7 of the calcitonin receptor favors Gs coupling over Gq.

Mutations in the thyrotropin receptor uncouple from Gq while maintaining coupling to Gs.

Related isoforms of some receptors (derived from alternative splicing of a single gene or generated after RNA editing) show different abilities to activate distinct G proteins.

CAMs represent a special class of structurally modified receptors. The conformations of CAMs are often regarded to reflect intermediate or even fully activated states of the wild-type receptor. However, it is quite possible that their conformation only approximates one of the active conformations of the wild-type receptor and that, when further activated by an agonist, their final conformation is also quite different from that of the fully activated wild-type receptor. Hence, the study of GPCR activation through the analysis of such CAMs presents severe limitations. A safe standpoint is that CAMs help our understanding of the structure of the inactive state, but give no clue to the interactions resulting in the ligand-induced active conformation(s).

Certain CAMs only activate a single signalling pathway among those ordinarily activated by the agonist. This may explain why:

• For the a1B-adrenergic receptor, it was found that a Cys-to-Phe mutation in TM3 constitutively activates the receptor when measuring the phospholipase C activity, but not when measuring the phospholipase A2 activity. Thus, such a mutation is likely to stabilize the receptor in a conformation that approximates one of the active conformations of the wild-type receptor.

• [Sar1,Ile4,Ile8]AngII can produce maximal inositol phosphate signalling through the CAM AT1a receptors (N111A and N111G). However, no internalization of these CAMs takes place, even in the presence of saturating concentrations of this ligand.

Different CAMs may display different conformations. This may explain why:

• They may be differentially phosphorylated and internalized although they convey a similar agonist-independent activity to the receptor. This has been observed for different CAMs of the a1B-receptor: phosphorylation and internalization still proceeds with A293E mutation but not with D142A mutation (Mhaouty-Kodja et al., 1999).

• [Bpa(2)]PTHrP(1-36) was a partial agonist for the wild-type parathyroid hormone/ parathyroid hormone-related peptide receptor and its T410P CAM, but it acted as an inverse agonist for the H223R CAM (Carter et al., 2001).

0 0

Post a comment