Cells integrate multiple incoming signals, and a response to one signal can depend upon the presence or intensity of others. Most often, acute responses to multiple signals are simply additive, either positively or negatively. Occasionally, however, the response to simultaneous stimuli is markedly greater than the sum of the responses to each stimulus alone. Such superadditive responses may be quantitatively modest, but marked synergism can essentially create a Boolean AND gate, or coincidence detector, with which a cell responds significantly only when two signals are present simultaneously. Superadditive responses are not frequent. In a recent large-scale screen for signaling interactions in macrophages, only about 1.5% of the ligand pairs that were tested displayed significant synergism [1
]. In some cases, mechanisms of cellular synergism are well understood. These include multiple phosphorylation events, coactivation by transcription factors, induction of synthesis of subsequently regulated proteins, etc. Positively cooperative binding of activating ligands can also create apparent synergism over a narrow range of concentrations as each ligand increases the affinity of the other [2
]. Scaffolding proteins and membrane surfaces potentiate signals essentially by this mechanism [5
]. For many acute superadditive cellular responses, however, mechanisms of synergism involve multiple signaling pathways, are otherwise complex [8
], or are unknown.
Here we use phospholipase C-β3 (PLC-β3) to elucidate general mechanisms for creating synergism through allosteric regulation, and we show that PLC-β3 regulation accounts for a well-known set of superadditive responses in diverse cells. It has been known for about 15 years that many animal cells and primary cell lines display synergistic Ca2+
responses to simultaneous inputs from different G protein-coupled receptors [10
]. In these cells, synergism serves as a coincidence detector, such that a robust Ca2+
response and downstream physiological regulation are only observed when both G protein pathways are activated. Such synergism is physiologically important in platelets, neurons, and macrophages [10
] and is suggested to play a role in stimulation of mitogenesis in multiple cell types [20
]. In most of these cases, one of the two receptors activates Gq
and the other activates Gi
, and synergism does not depend on which Gi
- or Gq
-coupled receptor initiates the signals. Gq
both activate PLC-β isoforms, and the PLC reaction product, inositol-trisphosphate (IP3
), triggers Ca2+
release from the endoplasmic reticulum to the cytosol [21
stimulates PLC-β via its Gαq
subunit, and Gi
acts via its Gβγ subunit [21
]. Several studies suggested that the mechanism of synergistic Ca2+
signaling directly involves PLC activation [10
], and recent studies in macrophages and a macrophage-like cell line argue that synergistic stimulation of Ca2+
signaling primarily requires the PLC-β3 isoform [10
]. However, other work suggested that cellular Gi
synergism involves interaction between the G proteins [25
] or the IP3
], and its biochemical mechanism remained unknown.
We show here that purified PLC-β3 responds synergistically to stimulation by Gαq and Gβγ. Synergistic activation of PLC-β3 can exceed ten times the sum of the responses to the individual G protein subunits. Gβγ-Gαq synergism on PLC-β3 can thus quantitatively account for synergistic Ca2+ responses to Gi and Gq in cells, and its biochemical behavior is qualitatively consistent with cellular events. Additional proteins or pathways are not required.
We also show that the synergistic response of PLC-β3 to Gαq and Gβγ can be explained quantitatively by a simple and classical two-state allosteric model. Synergism does not merely reflect positively cooperative effects of each subunit on the binding affinity of the other, but results from increased accumulation of the active form of PLC-β3. Synergism occur seven when both Gαq and Gβγ are tested at saturating concentrations.
The other PLC-β isoforms do not mediate synergistic Ca2+
responses in cells [10
] or display synergism in vitro, even though they are structurally homologous to PLC-β3 and respond similarly to individual G proteins [21
In general, why does one enzyme respond synergistically to two activators while another does not? We show by modeling and by analysis of PLC-β regulation that a superadditive response by a single enzyme primarily depends on its having very low activity in the absence of stimulating ligand. Maximal attainable synergism by a simple two-state enzyme is approximately proportional to its intrinsic bias for the inactive state. A two-state enzyme whose intrinsic activation is ≥ 1% of maximal cannot display more than two-fold synergism, and it can do so only with ligands that are fortuitously matched in their efficacies and that are at near perfect concentrations. In contrast, an enzyme with intrinsic activity ≤ 0.1% will display synergism to most activators and will do so over a broad range of activator concentrations. Thus any allosteric enzyme with a large dynamic range of regulation will display a synergistic response to two or more activating ligands. Synergism, which is widely assumed to be a complex phenomenon requiring ligand-ligand interactions or multiple activity states, can be described by a simple two-state allosteric equilibrium.