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Vol. 60, Issue 2, 321-330, August 2001
Department of Cell Physiology and Pharmacology, University of
Leicester, Leicester, UK (J.M.W., R.A.J.C., S.R.N.); and Department of
Pharmacology, University of Bristol, School of Medical Sciences,
Bristol, UK (E.K.)
We have investigated the effects of G protein-coupled receptor
kinase (GRK) 3 and GRK6 on the phosphorylation and regulation of the
M3 muscarinic acetylcholine receptor (mACh) endogenously expressed in SH-SY5Y cells. Overexpression of GRK3 or GRK6 enhanced M3 mACh receptor phosphorylation after high-concentration
methacholine (100 µM, 1 min) addition. However, GRK6 was more potent,
increasing receptor phosphorylation even after low (3 µM, 1 min)
agonist stimulation. Compared with plasmid-transfected control cells
expressing equivalent M3 mACh receptor number, GRK3- or
GRK6-overexpressing cells exhibited a reduced phospholipase C activity
reflected by a lower accumulation of total [3H]inositol
phosphates and Ins(1,4,5)P3 mass. In addition, direct stimulation of G protein activation of phospholipase C (by
AlF4
) was inhibited in GRK3- but not
GRK6-overexpressing cells.
Guanosine-5'-O-(3-[35S]thio)triphosphate
binding and immunoprecipitation of G
q/11 indicated that
acute methacholine-stimulated receptor/Gq/11 coupling was unaffected by GRK overexpression. In contrast, agonist pretreatment of cells for 3 min caused M3 mACh receptor uncoupling from
Gq/11, which was markedly enhanced by GRK6
overexpression, particularly at lower agonist pretreatment
concentrations. However, the increased M3 mACh receptor
phosphorylation seen in clones overexpressing GRK3 was not accompanied
by increased receptor-Gq/11 uncoupling. Overall, these
data suggest that GRK3 and GRK6 use different pathways to desensitize
the M3 mACh receptor. GRK6 seems to act as a classical GRK,
inducing increased receptor phosphorylation accompanied by an
uncoupling of receptor and Gq/11. Conversely, GRK3 may
cause desensitization independently of receptor phosphorylation,
possibly via G
binding and/or direct Gq binding
via its regulator of G protein signaling domain to inhibit
phospholipase C activity.
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