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Functional domains of the mouse β 3 ‐adrenoceptor associated with differential G‐protein coupling
Author(s) -
Sato Masaaki,
Hutchinson Dana S,
Evans Bronwyn A,
Summers Roger J
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.722.5
Subject(s) - pertussis toxin , receptor , gene isoform , g protein , mutagenesis , microbiology and biotechnology , chemistry , biology , mutation , biochemistry , gene
Alternative splicing of the mouse β 3 ‐adrenoceptor (AR) produces two isoforms of the receptor: the β 3a ‐AR couples to Gs and the β 3b ‐AR couples to both Gs and Gi. A truncated receptor that lacks the C‐terminus of the β 3a ‐ or the β 3b ‐AR showed pertussis toxin (PTX) sensitivity in a cyclic AMP (cAMP) accumulation assay, suggesting that the β 3a ‐AR is restrained from coupling to Gi by residues in the C‐terminus. This view was supported by studies using a cell‐penetrating peptide transportan‐10 (Tp10) coupled to the C‐terminal tail of the β 3a ‐ARτhat caused the β 3a ‐AR to become PTX sensitive. Site‐directed mutagenesis was used to identify residues in the C‐terminus of the β 3a ‐AR responsible for inhibition of coupling to Gi. Mutation of a putative caveolin binding site caused β 3a ‐AR mediated cAMP accumulation to become PTX sensitive (control, 1.00±0.16; PTX, 1.75±0.14, n=4, P<0.0001). Furthermore, treatment with filipin III, an agent that disrupts lipid rafts, also caused the wild type β 3a ‐AR to become PTX sensitive (control, 1.00±0.19; PTX, 1.56±0.04, n=4, P<0.0001), but in addition concentration‐response curves to CL316243 were markedly shifted to the right (pEC 50 control: 9.76±0.03, +filipin III: 8.73±0.21). These observations suggest that β 3a ‐ARs are restricted to caveolae and that localization of the receptor may play a specific role in G‐protein mediated signalling. This research was supported by National Health and Medical Research Council Project Grant 236884.