Аннотация
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.
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