Functional Coupling of the G_αolf Variant XLG_αolf with the Human Adenosine A_2A Receptor

Abstract

A recently identified novel Gα_olf variant, XLGα_olf, is shown to functionally couple to the human adenosine A_2A receptor (A_2AR). In Sf9 cells expressing A_2AR, β1, and γ2, co-expression of XLGα_olf increased NECA-induced [³⁵S]GTPγS binding from approximately 130% to 300% of basal levels. Pharmacological characteristics of A_2AR ligands on these cells were evaluated by using [³H]ZM241385- and [³⁵S]GTPγS- binding assays. The rank order of the equilibrium binding constants (K_d or K_i) of adenosine receptor ligands were [³H]ZM241385 ≈ CGS15943 < MRS1220 < < CV1808 ≈ NECA < CGS21680 ≈ adenosine < IBMECA < HEMADO ≈ CPA ≈ CCPA. The rank order of EC₅₀ values for agonists were CV1808 ≈ NECA < adenosine ≈ CGS26180 < IBMECA < HEMADO ≈ CPA ≈ CCPA. This pharmacology is consistent with the literature for A_2AR and suggests that Sf9 cells co-expressing A_2AR, β1, γ2, and XLGα_olf could serve as a heterologous expression system for A_2AR drug screening.

Key Words: :

• Adenosine receptor
• Gα_olf
• GTP_γ-S binding
• Insect cells
• HTS
• Screening
• G protein.

ABBREVIATIONS:
ADA	=	adenosine deaminase
CCPA	=	2-Chloro- N6-cyclopentyladenosine
CGS15943	=	9-Chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine
CGS21680	=	2- p-(2-carboxyethyl)phenethylamino-5′N-ethylcarboxamidoadenosine
CPA	=	N6-cyclopentyladenosine
CV1808	=	2-phenylaminoadenosine
GDP	=	guanosine diphosphate
GTPγS	=	guanosine5′-[γ-thio]triphosphate
HEMADO	=	2-(1-Hexynyl)-N-methyladenosine
MRS1220	=	N-[9-Chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzene
MRS1706	=	N-(4-Acetylphenyl)-2 [4[(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1Hpurin-8-yl)phenoxy]acetamide
NECA	=	N-ethylcarboxamidoadenosine
IB-MECA	=	1-deoxy-1-[6[(3-idophenyl)ethyl] amino-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide
DPCPX	=	8-cyclopentyl-1,3-dipropylxanthine
ZM241385	=	4-(2-[7-amino-2(2-furyl)[1,2,4]triazolo [2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol.

ABBREVIATIONS:
ADA	=	adenosine deaminase
CCPA	=	2-Chloro- N6-cyclopentyladenosine
CGS15943	=	9-Chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine
CGS21680	=	2- p-(2-carboxyethyl)phenethylamino-5′N-ethylcarboxamidoadenosine
CPA	=	N6-cyclopentyladenosine
CV1808	=	2-phenylaminoadenosine
GDP	=	guanosine diphosphate
GTPγS	=	guanosine5′-[γ-thio]triphosphate
HEMADO	=	2-(1-Hexynyl)-N-methyladenosine
MRS1220	=	N-[9-Chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzene
MRS1706	=	N-(4-Acetylphenyl)-2 [4[(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1Hpurin-8-yl)phenoxy]acetamide
NECA	=	N-ethylcarboxamidoadenosine
IB-MECA	=	1-deoxy-1-[6[(3-idophenyl)ethyl] amino-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide
DPCPX	=	8-cyclopentyl-1,3-dipropylxanthine
ZM241385	=	4-(2-[7-amino-2(2-furyl)[1,2,4]triazolo [2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol.