ABSTRACT
BACKGROUND: G protein-coupled receptors fused to a Gα-subunit are functionally similar to their unfused counterparts. They offer an intriguing view into the nature of the receptor-G protein complex, but their usefulness depends upon the stability of the fusion. METHODS: Fusion proteins of the M(2) muscarinic receptor and the α-subunit of G(i1) were expressed in CHO and Sf9 cells, extracted in digitonin-cholate, and examined for their binding properties and their electrophoretic mobility on western blots. RESULTS: Receptor fused to native α(i1) underwent proteolysis near the point of fusion to release a fragment with the mobility of α(i1). The cleavage was prevented by truncation of the α-subunit at position 18. Binding of the agonist oxotremorine-M to the stable fusion protein from Sf9 cells was biphasic, and guanylylimidodiphosphate promoted an apparent interconversion of sites from higher to lower affinity. With receptor from CHO cells, the apparent capacity for N-[(3)H]methylscopolamine was 60% of that for [(3)H]quinuclidinylbenzilate; binding at saturating concentrations of the latter was inhibited in a noncompetitive manner at low concentrations of unlabeled N-methylscopolamine. CONCLUSIONS: A stable fusion protein of the M(2) receptor and truncated α(i1) resembles the native receptor-G protein complex with respect to the guanyl nucleotide-sensitive binding of agonists and the noncompetitive binding of antagonists. GENERAL SIGNIFICANCE: Release of the α-subunit is likely to occur with other such fusion proteins, rendering the data ambiguous or misleading. The properties of a chemically stable fusion protein support the notion that signaling proceeds via a stable multimeric complex of receptor and G protein.
Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , Ligands , Receptor, Muscarinic M2/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Amino Acid Sequence , Animals , CHO Cells , Cell Line , Cells, Cultured , Cricetinae , Cricetulus , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Humans , Molecular Sequence Data , Protein Binding , Receptor, Muscarinic M2/metabolism , Sequence AlignmentABSTRACT
Muscarinic receptor extracted from porcine atria in digitonin-cholate copurified with Galpha(o), Galpha(i1-3), and caveolins. The presence of complexes was confirmed by coimmunoprecipitation of the receptor, alpha-subunits, and caveolins in various combinations. Homooligomers of alpha(i2) were detected on Western blots, and heterooligomers of alpha(i2) and alpha(o) were identified by coimmunoprecipitation; thus, a complex may contain at least two alpha-subunits. Other combinations of alpha-subunit were not detected. The ratio of total alpha-subunit to receptor was near 1, as measured by [(35)S]GTPgammaS and the antagonist [(3)H]quinuclidinylbenzilate, and the binding of [(35)S]GTPgammaS was manifestly biphasic. The ratio of alpha(o) to alpha(i1,2) also was near 1, as determined from the intensity of Western blots. Cardiac muscarinic receptors therefore can be purified as a mixture of complexes that contain caveolins and oligomers of alpha-subunit, some of which are heteromeric. Each complex would appear to contain equal numbers of alpha-subunit and the receptor.
Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Heart Atria/metabolism , Receptor, Muscarinic M2/metabolism , Swine/metabolism , Animals , Caveolins/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Muscarinic Antagonists/pharmacology , Protein Subunits/isolation & purification , Protein Subunits/metabolism , Quinuclidinyl Benzilate/pharmacology , Receptor, Muscarinic M2/antagonists & inhibitors , Receptor, Muscarinic M2/isolation & purificationABSTRACT
FLAG- and HA-tagged M2 muscarinic receptors from coinfected Sf9 cells have been purified in digitonin-cholate and reconstituted into phospholipid vesicles. The purified receptor was predominantly monomeric: it showed no detectable coimmunoprecipitation; it migrated as a monomer during electrophoresis before or after cross-linking with bis(sulfosuccinimidyl)suberate; and it bound agonists and antagonists in a manner indicative of identical and mutually independent sites. Receptor cross-linked after reconstitution or after reconstitution and subsequent solubilization in digitonin-cholate migrated almost exclusively as a tetramer. The binding properties of the reconstituted receptor mimicked those reported previously for cardiac muscarinic receptors. The apparent capacity for N-[3H]methylscopolamine (NMS) was only 60% of that for [3H]quinuclidinylbenzilate (QNB), yet binding at saturating concentrations of [3H]QNB was inhibited fully and in a noncompetitive manner at comparatively low concentrations of unlabeled NMS. Reconstitution of the receptor with a saturating quantity of functional G proteins led to the appearance of three classes of sites for the agonist oxotremorine-M in assays with [3H]QNB; GMP-PNP caused an apparent interconversion from highest to lowest affinity and the concomitant emergence of a fourth class of intermediate affinity. All of the data can be described quantitatively in terms of cooperativity among four interacting sites, presumably within a tetramer; the effect of GMP-PNP can be accommodated as a shift in the distribution of tetramers between two states that differ in their cooperative properties. Monomers of the M2 receptor therefore can be assembled into tetramers with binding properties that closely resemble those of the muscarinic receptor in myocardial preparations.
