Long-term agonist stimulation of IP prostanoid receptor depletes the cognate Gsα protein in membrane domains but does not change the receptor level

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Abstract

Iloprost (IP) stimulation (1 μM, 2 h) of Flag-epitope-tagged human IP prostanoid receptor (FhIPR) expressed in HEK293 cells resulted in specific decrease of endogenous Gsα protein in detergent-insensitive, caveolin-enriched, membrane domains (DIMs). Receptor protein FhIPR, caveolin, Giα and GPI-linked, domain markers CD55 and CD59 were unchanged. The same result was obtained in HEK293 cells expressing FhIPR-Gsα fusion protein. The endogenous Gsα decreased, but the level of Flag-hIPR-Gsα protein did not change. The specific depletion of domain-bound pool of Gsα as consequence of iloprost stimulation was also demonstrated in membrane domains prepared according to alkaline treatment plus sonication protocol (detergent-free procedure of Song et al. [J. Biol. Chem. 271 (1996) 9690]). Our data further indicated that in control, quiescent cells only a very small amount of IP prostanoid receptor was present in DIMs together with large amount of its cognate Gsα protein. Expressed in quantitative terms, DIMs contained 30–40% of the total cellular amount of G proteins whereas the content of IP prostanoid receptors was 1–3%. The dominant portion (>95%) of FhIPR as well as FhIPR-Gsα was localised in high-density area of the gradient containing detergent-solubilised proteins. FhIPR and FhIPR-Gsα distribution was similar to that of transmembrane plasma membrane (PM) markers (CD147, MHCI, CD29, Tapa1, the α subunit of Na,K-ATPase, transmembrane form of CD58 and CD44). All these proteins are known to be fully solubilised by detergent and thus unable to float in density gradient.

Our data indicate that (i) long-term agonist stimulation of IP prostanoid receptor is associated with preferential decrease of its cognate G protein Gsα from membrane domains; receptor level is unchanged. (ii) Very small fraction (1–3%) of total cellular amount of receptors is recovered in DIMs together with roughly 40% of G proteins. These data suggest a “supra-stoichiometric” arrangement of G proteins and corresponding receptors in DIMs.

Keywords

IP prostanoid receptor
Gsα protein
Long-term agonist stimulation
Membrane domain

Abbreviations

BPB
bromphenol blue
DIMs
detergent-insoluble or detergent-insensitive membrane domains
DMEM
Dulbecco's modified Eagle's medium
FLAG
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
FhIPR
Flag-tagged form of human IP prostanoid receptor
FhIPR-Gsα
FhIPR-Gsα fusion protein
G proteins
heterotrimeric guanine nucleotide-binding regulatory proteins
Gsα
G protein stimulating adenylyl cyclase activity
Giα
G proteins inhibiting adenylyl cyclase activity in pertussis-toxin sensitive manner
Gqα/G11α
G proteins stimulating phosholipase C in pertussis-toxin independent manner
GPCR
G protein coupled receptor
HEK
human embryonic kidney
GPI
glycosylphosphatidylinositol
Mr
relative molecular weight
PBS
phosphate-buffered saline
PM
plasma membranes
PMSF
phenylmethylsulfonyl fluoride
Na,K-ATPase
sodium plus potassium activated, ouabain dependent adenosine triphosphatase (EC 3.6.1.3)
SLB
solubilisation lysis buffer
TBS
Tris-buffered saline
TRH
thyrotropin releasing hormone
TRH-R
TRH receptor

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