Abstract
The muscarinic M3 receptor (M3R)
is a Gq-coupled receptor and is
known to interact with many intracellular regulatory proteins. One
of these molecules is Gβ5-RGS7, the permanently associated heterodimer
of G protein β-subunit Gβ5 and RGS7, a regulator of G
protein signaling. Gβ5-RGS7 can attenuate M3R-stimulated release
of Ca
2+
from intracellular stores or enhance the influx
of Ca
2+
across the plasma membrane. Here we show that deletion
of amino acids 304–345 from the central portion of the i3 loop
renders M3R insensitive to regulation by Gβ5-RGS7. In addition
to the i3 loop, interaction of M3R with Gβ5-RGS7 requires helix
8. According to circular dichroism spectroscopy, the peptide corresponding
to amino acids 548–567 in the C-terminus of M3R assumes an
α-helical conformation. Substitution of Thr553 and Leu558 with
Pro residues disrupts this α-helix and abolished binding to
Gβ5-RGS7. Introduction of the double Pro substitution into full-length
M3R (M3R
TP/LP
) prevents trafficking of the receptor to
the cell surface. Using atropine or other antagonists as pharmacologic
chaperones, we were able to increase the level of surface expression
of the TP/LP mutant to levels comparable to that of wild-type M3R.
However, M3R-stimulated calcium signaling is still severely compromised.
These results show that the interaction of M3R with Gβ5-RGS7
requires helix 8 and the central portion of the i3 loop.
