Abstract
The studies reported here address the molecular events underlying the interactions of arrestins with the M(2) muscarinic acetylcholine receptor (mAChR). In particular, we focused on the role of receptor phosphorylation in this process. Agonist-dependent phosphorylation of the M(2) mAChR can occur at clusters of serines and threonines at positions 286-290 (site P1) or 307-311 (site P2) in the third intracellular loop (Pals-Rylaarsdam, R., and Hosey, M. M. (1997) J. Biol. Chem. 272, 14152-14158). Phosphorylation at either P1 or P2 can support agonist-dependent internalization. However, phosphorylation at P2 is required for receptor interaction with arrestins (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682-26389). The present study investigated the role of acidic amino acids between P1 and P2 in regulating receptor phosphorylation, internalization, and receptor/arrestin interactions. Mutation of the acidic amino acids at positions 298-300 (site A1) and/or 304-305 (site A2) to alanines had significant effects on agonist-dependent phosphorylation. P2 was identified as the preferred site of agonist-dependent phosphorylation, and full phosphorylation at P2 required the acidic amino acids at A1 or their neutral counterparts. In contrast, phosphorylation at site P1 was dependent on site A2. In addition, sites A1 and A2 significantly affected the ability of the wild type and P1 and P2 mutant receptors to internalization and to interact with arrestin2. Substitution of asparagine and glutamine for the aspartates and glutamates at sites A1 or A2 did not influence receptor phosphorylation but did influence arrestin interaction with the receptor. We propose that the amino acids at sites A1 and A2 play important roles in agonist-dependent phosphorylation at sites P2 and P1, respectively, and also play an important role in arrestin interactions with the M(2) mAChR.
Highlights
Since the P2 mutant of the M2 muscarinic acetylcholine receptor (mAChR) was unable to bind to phosphorylation-independent forms of arrestin, these results suggested that different molecular events participate in the interaction of arrestins with the M2 mAChR compared with those governing the interactions of arrestins with Rh or the 2AR
These results indicated that mutation of the acidic amino acids between the P1 and P2 clusters influenced the ability of the M2 mAChR to interact with arrestin2
Effect of Conservative Mutations on the Acidic Amino Acids Affecting Arrestin-promoted Internalization—In order to investigate if the chemical/steric nature of the acidic amino acids participated in receptor interactions with arrestin, we studied the receptors in which the aspartic and glutamic acids of A1 and A2 were conservatively mutated to asparagine and glutamine in the wild type M2 mAChR background (WT*A1QN or WT*A2QN mutants, see Fig. 1)
Summary
Materials—Dulbecco’s modified Eagle’s medium (DMEM) and penicillin/streptomycin were purchased from Mediatech. Creation of the A1 and A2 receptor mutants, as well as the WT*A1QN and WT*A2QN receptor mutants (Fig. 1B), was performed by mutating amino acids 298 –300 or 304 –305 in the vector constructs of the wild type (M2 mAChR pCR3), *P1 (M2 Nala-4 mAChR pCR3 [35]), and *P2 (M2 Cala-4 mAChR pCR3 [35]) using the QuickChange sitedirected mutagenesis kit (Stratagene) as described by the manufacturer’s instructions. Phosphorylation Assay in Intact Cells—HEK-tsA201 cells transiently expressing the various receptor constructs were incubated in phosphate-free DMEM with 400 Ci of 32Pi for a minimum of 4 h and incubated at room temperature for 20 min in the presence or absence of 1 mM carbachol. Immunoblot Analysis—The expression of arrestin, dominant-negative arrestin (arrestin3-(293– 405)), or hemagglutinin-tagged dominant-negative dynamin (hemagglutinin-dynK44A) was analyzed by Western blotting as described previously [39]
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