Phospholipase C-β (PLCβ) is a key regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to Gq. We have determined atomic structures of two invertebrate homologs of PLCβ (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCβ3 dramatically increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCβ.
G protein-coupled receptors (GPCRs) have been found as monomers but also as dimers or higher-order oligomers in cells. The relevance of the monomeric or dimeric receptor state for G protein activation is currently under debate for class A rhodopsin-like GPCRs. Clarification of this issue requires the availability of well defined receptor preparations as monomers or dimers and an assessment of their ligand-binding and G protein-coupling properties. We show by pharmacological and hydrodynamic experiments that purified neurotensin receptor NTS1, a class A GPCR, dimerizes in detergent solution in a concentration-dependent manner, with an apparent affinity in the low nanomolar range. At low receptor concentrations, NTS1 binds the agonist neurotensin with a Hill slope of Ϸ1; at higher receptor concentrations, neurotensin binding displays positive cooperativity with a Hill slope of Ϸ2. NTS1 monomers activate G␣q 1␥2, whereas receptor dimers catalyze nucleotide exchange with lower affinity. Our results demonstrate that NTS1 dimerization per se is not a prerequisite for G protein activation.dimer ͉ G protein activation ͉ G protein-coupled receptor ͉ monomer D imerization of G protein-coupled receptors (GPCRs) has been the subject of intense interest. Class C GPCRs, such as metabotropic glutamate receptors and ␥-aminobutyric acid type B (GABA B ) receptors, clearly form homo-and heterodimeric structures, essential both for trafficking of receptors to the cell surface and for ligand-induced activation of receptors and G protein coupling. Detailed models for receptor and G protein activation have been proposed that account for the multidomain structure of class C GPCRs (for review, see ref. 1).In contrast, no conclusion has yet been reached as to the importance of dimerization for class A receptor function; the role of receptor monomers (2) or dimers (3, 4) in signal transduction is controversial. Models proposed to explain the mechanism of receptor-catalyzed G protein activation (5-7) assumed the interaction of G protein with a receptor monomer. More recently, class A GPCRs have been described as monomers (8) and dimers (see refs. 9 and 10) in living cells by resonance energy transfer methods. For rhodopsin, dimer particles and higher-order oligomers have been visualized in disk membranes by atomic force microscopy (11). Based on structural constraints, a model was suggested in which a receptor dimer provides an extensive contact area for the heterotrimeric G protein; the surface area of a GPCR monomer was deemed insufficiently large to anchor both the G␣ and G␥ subunits simultaneously (3, 4, 12). However, alternate models for the interaction of a monomeric rhodopsin with a G protein heterotrimer have also been proposed (2).The concentration of rhodopsin in rod outer segment disk membranes is high (Ϸ2.5 mM; for review, see ref. 13), and rhodopsin may therefore exist only as dimers, as seen by atomic force microscopy (11). Because a rod cell can respond to a single photon (14, 15), only one activated rhodopsin protomer, w...
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