### A. Overview

Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface receptors termed purine receptors. In this review particular emphasis is placed on the discrepancy between the

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Receptors for Purines and Pyrimidines

Seven-transmembrane receptors, which constitute the largest, most ubiquitous and most versatile family of membrane receptors, are also the most common target of therapeutic drugs. Recent findings indicate that the classical models of G-protein coupling and activation of second-messenger-generating enzymes do not fully explain their remarkably diverse biological actions.

Seven-transmembrane receptors.

G protein-coupled receptors (GPCRs) are seven transmembrane proteins that form the largest single family of integral membrane receptors. GPCRs transduce information provided by extracellular stimuli into intracellular second messengers via their coupling to heterotrimeric G proteins and the subsequent regulation of a diverse variety of effector systems. Agonist activation of GPCRs also initiates processes that are involved in the feedback desensitization of GPCR responsiveness, the internalization of GPCRs, and the coupling of GPCRs to heterotrimeric G protein-independent signal transduction pathways. GPCR desensitization occurs as a consequence of G protein uncoupling in response to phosphorylation by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes the binding of beta-arrestins, which not only uncouple receptors from heterotrimeric G proteins but also target many GPCRs for internalization in clathrin-coated vesicles. beta-Arrestin-dependent endocytosis of GPCRs involves the direct interaction of the carboxyl-terminal tail domain of beta-arrestins with both beta-adaptin and clathrin. The focus of this review is the current and evolving understanding of the contribution of GRKs, beta-arrestins, and endocytosis to GPCR-specific patterns of desensitization and resensitization. In addition to their role as GPCR-specific endocytic adaptor proteins, beta-arrestins also serve as molecular scaffolds that foster the formation of alternative, heterotrimeric G protein-independent signal transduction complexes. Similar to what is observed for GPCR desensitization and resensitization, beta-arrestin-dependent GPCR internalization is involved in the intracellular compartmentalization of these protein complexes.

Evolving Concepts in G Protein-Coupled Receptor Endocytosis: The Role in Receptor Desensitization and Signaling

Eleven distinct isoforms of phosphoinositide-specific phospholipase C (PLC), which are grouped into four subfamilies (beta, gamma, delta, and epsilon), have been identified in mammals. These isozymes catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to inositol 1,4,5-trisphosphate and diacylglycerol in response to the activation of more than 100 different cell surface receptors. All PLC isoforms contain X and Y domains, which form the catalytic core, as well as various combinations of regulatory domains that are common to many other signaling proteins. These regulatory domains serve to target PLC isozymes to the vicinity of their substrate or activators through protein-protein or protein-lipid interactions. These domains (with their binding partners in parentheses or brackets) include the pleckstrin homology (PH) domain [PtdIns(3)P, beta gamma subunits of G proteins] and the COOH-terminal region including the C2 domain (GTP-bound alpha subunit of Gq) of PLC-beta; the PH domain [PtdIns(3,4,5)P3] and Src homology 2 domain [tyrosine-phosphorylated proteins, PtdIns(3,4,5)P3] of PLC-gamma; the PH domain [PtdIns(4,5)P2] and C2 domain (Ca2+) of PLC-delta; and the Ras binding domain (GTP-bound Ras) of PLC-epsilon. The presence of distinct regulatory domains in PLC isoforms renders them susceptible to different modes of activation. Given that the partners that interact with these regulatory domains of PLC isozymes are generated or eliminated in specific regions of the cell in response to changes in receptor status, the activation and deactivation of each PLC isoform are likely highly regulated processes.

Regulation of phosphoinositide-specific phospholipase C

The central role of phosphoinositide 3-kinase (PI3K) activation in tumour cell biology has prompted a sizeable effort to target PI3K and/or downstream kinases such as AKT and mammalian target of rapamycin (mTOR) in cancer. However, emerging clinical data show limited single-agent activity of inhibitors targeting PI3K, AKT or mTOR at tolerated doses. One exception is the response to PI3Kδ inhibitors in chronic lymphocytic leukaemia, where a combination of cell-intrinsic and -extrinsic activities drive efficacy. Here, we review key challenges and opportunities for the clinical development of inhibitors targeting the PI3K-AKT-mTOR pathway. Through a greater focus on patient selection, increased understanding of immune modulation and strategic application of rational combinations, it should be possible to realize the potential of this promising class of targeted anticancer agents.

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PI3K and cancer: lessons, challenges and opportunities

Characterization of an altered membrane form of the beta-adrenergic receptor produced during agonist-induced desensitization.

Beta gamma-subunit activation of G-protein-regulated phospholipase C.

Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C (PLC)–dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide–binding proteins (G proteins) activate PLC-βs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure of PLC-β3 bound to activated Gαq reveals a conserved module found within PLC-βs and other effectors optimized for rapid engagement of activated G proteins. The active site of PLC-β3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein–dependent anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-β3 subsequently accelerates guanosine triphosphate hydrolysis by Gαq, causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal signals mediated by diverse sensory inputs.

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Kinetic Scaffolding Mediated by a Phospholipase C–β and Gq Signaling Complex

RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of Gi family G-proteins with differential selectivity and maximal activity.

Incubation of astrocytoma cells with catecholamines results in a decrease in catecholamine-stimulated adenylate cyclase activity and a concomitant alteration in the sedimentation properties of particulate beta-adrenergic receptors The altered receptors exhibit agonist binding properties similar to those of receptors that are "uncoupled" from adenylate cyclase

G L Waldo

Papers

Characterization of an altered membrane form of the beta-adrenergic receptor produced during agonist-induced desensitization.

Beta gamma-subunit activation of G-protein-regulated phospholipase C.

Kinetic Scaffolding Mediated by a Phospholipase C–β and Gq Signaling Complex

RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of Gi family G-proteins with differential selectivity and maximal activity.

Catecholamine-induced alteration in sedimentation behavior of membrane bound beta-adrenergic receptors