Quantitative Description of Signaling Downstream of Gq-Coupled Receptors: Similarities and Differences in the Responses of IP3, Calcium, DAG, PKC, and PIP2

Abstract

Gq-coupled plasma membrane receptors modulate cellular functions by activating phospholipase C (PLC), which hydrolyses the membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) into the second messengers inositoltrisphosphate (IP3) and diacylgycerol (DAG). To better understand the mechanisms that govern these partially independent signals we monitored in single, living tsA-201 cells levels of PIP2, IP3, calcium, DAG, and PKC by optical probes and current. We compared (i) activation of (low-abundance) endogenous purinergic receptors and overexpressed M1 muscarinic receptors, and (ii) different concentrations of the muscarinic agonist oxotremorine-M (oxo-M). Whereas the peak responses from reporters of IP3 (LIBRAvIII) and DAG (C1 domains of PKCγ) scale with abundance of receptor or agonist, downstream production of calcium (Fura4F) and PKC activation (CKAR) do not. Amplitude and duration of calcium signals elicited by 100 µM UTP, 10 nM oxo-M, or 10 µM oxo-M are almost identical. The only difference is a shorter latency with 10 µM oxo-M. These data suggest that a relatively low amount of IP3 is required for calcium release. This interpretation is supported by the finding that a full-size calcium response can still be elicited after PIP2 is depleted by recruiting a PI 5-phosphatase to the plasma membrane (by rapamycin-induced dimerization). Duration and late recovery time courses are different between IP3 (duration=68 s; τoff =55 s) and calcium (duration=110 s; τoff =34 s), suggesting that once a threshold of IP3 is reached, the calcium signal unfolds. Therefore we conclude that the IP3 requirement for calcium release must be low. The time point and IP3 level (from LIBRAvIII) at which the calcium response starts can provide an estimate of this IP3 threshold. Supported by NIH grants NS08174 & GM83913 and the HFSP.