Homologous and heterologous uncoupling of muscarinic M 3 and α 1B adrenoceptors to Gα q/11 in SH‐SY5Y human neuroblastoma cells

• Published in: British journal of pharmacology Vol. 134; no. 2; pp. 257 - 264
• Main Authors: Bundey, R A, Nahorski, S R
• Format: Journal Article
• Language: English
• Published: 02-02-2009
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0704229

The present study employed a [
35
S]‐GTPγS binding protocol in conjunction with immunoprecipitation (IP) of the Gα subunits to investigate the desensitization of G
q/11
‐coupled receptors at the level of the G‐protein activation. Membranes from SH‐SY5Y cells expressing the recombinant human α
1B
‐adrenoceptor (α
1B
‐AR) (and endogenously expressing the M
3
muscarinic acetylcholine receptor (M
3
‐AChR)) exhibited G
q/11
activation in a concentration‐dependent manner in response to noradrenaline or methacholine.
Pre‐treatment of intact cells with agonist prior to membrane preparation and use in the [
35
S]‐GTPγS IP assay demonstrated that both receptors were homologously desensitized by pre‐treatment with agonist since the G
q/11
activation in response to a secondary challenge with agonist was markedly reduced. Stimulation of α
1B
‐AR was effective at heterologously desensitizing the M
3
‐AChR. The PKC inhibitor, Ro‐31‐8220 (10 μ
M
) was ineffective at preventing the agonist‐mediated receptor desensitization.
[
32
P]P
i
‐labelled cells allowed the detection of increases in receptor phosphorylation. Phorbol 12,13 dibutyrate (PDBu) (1 μ
M
) was effective at producing a Ro‐31‐8220 (10 μ
M
)‐sensitive, detectable increase in α
1B
‐AR but not M
3
‐AChR phosphorylation. Noradrenaline (30 μ
M
) stimulated α
1B
‐AR phosphorylation, which could be partially inhibited by Ro‐31‐8220 (10 μ
M
). The phosphorylation of M
3
‐AChR was increased by methacholine (100 μ
M
) incubation and this effect appeared to be insensitive to Ro‐31‐8220 (10 μ
M
).
These findings demonstrate that [
35
S]‐GTPγS‐Gα‐subunit IP can be used to estimate receptor desensitization as a decline in receptor‐G‐protein coupling. Both the α
1B
‐AR and M
3
‐AChR undergo rapid homologous desensitization that is associated with an increase in receptor phosphorylation. The heterologous desensitization of M
3
‐AChR produced by α
1B
‐AR stimulation is not associated with a detectable increase in M
3
‐AChR phosphorylation, suggesting that receptor phosphorylation is not necessarily a prerequisite for desensitization.
British Journal of Pharmacology
(2001)
134
, 257–264; doi:
10.1038/sj.bjp.0704229