Experimental verification of the kinetic theory of FRET using optical microspectroscopy and obligate oligomers.

Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photo-insensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells.

Oligomeric size of the m2 muscarinic receptor in live cells as determined by quantitative fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M(2) muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M(2) receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M(2) receptor to enhanced green fluorescent protein-M(2) receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n = 2, 0.495 +/- 0.019; n = 4, 0.202 +/- 0.010; n = 6, 0.128 +/- 0.006; n = 8, 0.093 +/- 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20-0.24, identifying the M(2) receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process.

Binding of orthosteric ligands to the allosteric site of the M(2) muscarinic cholinergic receptor.

The M(2) muscarinic receptor has two topographically distinct sites: the orthosteric site and an allosteric site recognized by compounds such as gallamine. It also can exhibit cooperative effects in the binding of orthosteric ligands, presumably to the orthosteric sites within an oligomer. Such effects would be difficult to interpret, however, if those ligands also bound to the allosteric site. Monomers of the hemagglutinin (HA)- and FLAG-tagged human M(2) receptor therefore have been purified from coinfected Sf9 cells and examined for any effect of the antagonist N-methyl scopolamine or the agonist oxotremorine-M on the rate at which N-[(3)H]methyl scopolamine dissociates from the orthosteric site (k(obsd)). The predominantly monomeric status was confirmed by coimmunoprecipitation and by cross-linking with bis(sulfosuccinimidyl)suberate. Both N-methyl scopolamine and oxotremorine-M acted in a cooperative manner to decrease k(obsd) by 4.5- and 9.1-fold, respectively; the corresponding estimates of affinity (log K(L)) are -2.55 +/- 0.13 and -2.29 +/- 0.14. Gallamine and the allosteric ligand obidoxime decreased k(obsd) by more than 100-fold (log K(L) = -4.12 +/- 0.04) and by only 1.1-fold (log K(L) = -1.73 +/- 0.91), respectively. Obidoxime reversed the effect of N-methyl scopolamine, oxotremorine-M, and gallamine in a manner that could be described by a model in which all four ligands compete for a common allosteric site. Ligands generally assumed to be exclusively orthosteric therefore can act at the allosteric site of the M(2) receptor, albeit at comparatively high concentrations.

Cholesterol as a determinant of cooperativity in the M2 muscarinic cholinergic receptor.

M2 muscarinic receptor extracted from Sf9 cells in cholate-NaCl differs from that extracted from porcine sarcolemma. The latter has been shown to exhibit an anomalous pattern in which the capacity for N-[3H]methylscopolamine (NMS) is only 50% of that for [3H]quinuclidinylbenzilate (QNB), yet unlabeled NMS exhibits high affinity for all of the sites labeled by [3H]QNB. The effects can be explained in terms of cooperativity within a receptor that is at least tetravalent [Park PS, Sum CS, Pawagi AB, Wells JW. Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors. Biochemistry 2002;41:5588-604]. In contrast, M2 receptor extracted from Sf9 membranes exhibited no shortfall in the capacity for [3H]NMS at either 30 or 4 degrees C, although there was a time-dependent inactivation during incubation with [3H]NMS at 30 degrees C; also, any discrepancies in the affinity of NMS were comparatively small. The level of cholesterol in Sf9 membranes was only 4% of that in sarcolemmal membranes, and it was increased to about 100% by means of cholesterol-methyl-beta-cyclodextrin. M2 receptors extracted from treated Sf9 membranes were stable at 30 and 4 degrees C and resembled those from heart. Cholesterol induced a marked heterogeneity detected in the binding of both radioligands, including a shortfall in the apparent capacity for [3H]NMS, and there were significant discrepancies in the apparent affinity of NMS as estimated directly and via the inhibition of [3H]QNB. The data can be described quantitatively in terms of cooperative effects among six or more interacting sites. Cholesterol therefore appears to promote cooperativity in the binding of antagonists to the M2 muscarinic receptor.

Recovery of oligomers and cooperativity when monomers of the M2 muscarinic cholinergic receptor are reconstituted into phospholipid vesicles.

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.