Abstract
Fusion proteins were constructed between the alpha(2A)-adrenoceptor and the alpha-subunit of the G-protein G(i1). Mutation of the highly conserved Asp(79) in transmembrane (TM) helix 2 of the receptor to Asn reduced the capacity of agonists to activate G(i1)alpha by 95% without altering [3H]antagonist or agonist ligand-binding affinity. A reciprocal mutation in TM helix 7 (Asn(422)Asp) was without effect on signalling effectiveness. Combination of these two mutations overcame the effect of the Asp(79)Asp mutation. By examining alterations in this helix 2-helix 7 microdomain, we further demonstrate the utility of receptor-G-protein fusion proteins to quantitate mutational effects on receptor-G-protein interactions and information transfer.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Adrenergic alpha-2 Receptor Agonists*
-
Adrenergic alpha-Agonists / metabolism
-
Adrenergic alpha-Agonists / pharmacology*
-
Animals
-
Asparagine / genetics
-
Aspartic Acid / genetics
-
COS Cells
-
Conserved Sequence
-
Epinephrine / metabolism
-
Epinephrine / pharmacology
-
GTP-Binding Protein alpha Subunits, Gi-Go / genetics
-
GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
-
Guanosine 5'-O-(3-Thiotriphosphate) / pharmacology
-
Isoquinolines / metabolism
-
Mutation
-
Naphthyridines / metabolism
-
Protein Structure, Tertiary
-
Receptors, Adrenergic, alpha-2 / chemistry*
-
Receptors, Adrenergic, alpha-2 / genetics
-
Receptors, Adrenergic, alpha-2 / metabolism
-
Recombinant Fusion Proteins / metabolism
Substances
-
Adrenergic alpha-2 Receptor Agonists
-
Adrenergic alpha-Agonists
-
Isoquinolines
-
Naphthyridines
-
RS 79948-197
-
Receptors, Adrenergic, alpha-2
-
Recombinant Fusion Proteins
-
Aspartic Acid
-
Guanosine 5'-O-(3-Thiotriphosphate)
-
Asparagine
-
GTP-Binding Protein alpha Subunits, Gi-Go
-
Epinephrine