Some antagonists exhibit tissue selectivity in their pharmacological antagonism of muscarinic responses. However, the affinity constants for equilibrium binding of classical antagonists to muscarinic receptors in subcellular preparations have shown only small variations in different peripheral tissues and regions of the brain. The binding curves do not deviate significantly from the simple Langmuir isotherm, indicating apparent homogeneity of the receptor population in any given region. In contrast, heterogeneity has been detected by agonist binding studies but this may arise from different environmental or coupling restraints on the agonist-induced conformational change and cannot be taken as evidence for different receptor subtypes. We report here binding studies using a new anti-muscarinic drug, pirenzepine, in which we found heterogeneity of binding that correlates well with the pharmacological activity.

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Previously we identified MIR16 (membrane interacting protein of RGS16) as an integral membrane glycoprotein that interacts with regulator of G protein signaling proteins and shares significant sequence homology with bacterial glycerophosphodiester phosphodiesterases (GDEs), suggesting that it is a putative mammalian GDE. Here we show that MIR16 belongs to a large, evolutionarily conserved family of GDEs with a characteristic putative catalytic domain that shares a common motif (amino acids 92-116) with the catalytic domains of mammalian phosphoinositide phospholipases C. Expression of wild-type MIR16 (renamed GDE1), but not two catalytic domain mutants (E97A͞D99A and H112A), leads to a dramatic increase in glycerophosphoinositol phosphodiesterase (GPI-PDE) activity in HEK 293T cells. Analysis of substrate specificity shows that GDE1͞MIR16 selectively hydrolyzes GPI over glycerophosphocholine. The GPI-PDE activity of GDE1͞MIR16 expressed in HEK 293T cells can be regulated by stimulation of G proteincoupled, ␣͞␤-adrenergic, and lysophospholipid receptors. Membrane topology studies suggest a model in which the catalytic GDE domain faces the lumen͞extracellular space and the C terminus faces the cytoplasm. Our results suggest that by serving as a PDE for GPI with its activity regulated by G protein signaling, GDE1͞ MIR16 provides a link between phosphoinositide metabolism and G protein signal transduction.phosphoinositide ͉ RGS proteins ͉ phospholipid metabolism ͉ GDE domain T he glycerophospholipids, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidlylserine, are the major lipids of biological membranes. Of these, phosphatidylinositol (PtdIns) and its derivatives have taken on increasing importance based on their evolving roles as regulatory molecules in signal transduction and membrane trafficking during protein secretion, endocytosis, and cytoskeleton organization (1-4). Therefore, their metabolism and fate are of increasing interest. It is known that PtdIns, as well as other glycerophospholipids, can be deacylated to water-soluble glycerophosphodiesters (GPs) sequentially by phospholipase A and lysophospholipase or by phospholipase B alone (2, 5, and 6) and that GPs can be further hydrolyzed to sn-glycerol 3-phosphate and the corresponding alcohols by GP phosphodiesterases (GDEs). GDE activity toward glycerophosphoinositol (GPI), glycerophosphocholine (GPC), and glycerophosphoethanolamine (GPE) has been found in many mammalian tissues (7-11). Of these activities, glycerophosphoinositol phosphodiesterase (GPI-PDE) is of particular interest because of its participation in phosphoinositide (PI) metabolism.Recently we identified MIR16 (membrane interacting protein of RGS16) and showed that it binds to RGS16, as well as several other regulator of G protein signaling (RGS) proteins that serve as GTPase activating proteins (GAPs) for trimeric G proteins (12). We further showed that rat MIR16 is an integral membrane glycoprotein and shares significant sequence homology with bacterial GD...

New efforts in cancer therapy are being focused at various levels of signaling pathways. With phosphoinositide 3-kinase (PI3-K) potentially being necessary for a range of cancer-related functions, we have investigated the influence of selected inositol tris- to hexakisphosphates on cell growth and tumorigenicity. We show that micromolar concentrations of inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P(4)] inhibit IGF-1-induced [(3)H]-thymidine incorporation in human breast cancer (MCF-7) cells and the ability to grow in liquid medium and form colonies in agarose semisolid medium by small cell lung cancer (SCLC) cells, a human cancer cell line containing a constitutively active PI3-K. In an ovarian cancer cell line that also contains a constitutively active PI3-K (SKOV-3 cells), Ins(1,4,5,6)P(4) again inhibited liquid medium growth. Furthermore, when applied extracellularly, inositol 1,3,4,5-tetrakisphosphate was shown indeed to enter SCLC cells. These effects appeared specifically related to PH domains known to bind to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)], indicating involvement of the PI3-K downstream target protein kinase B (PKB/Akt). This was further supported by inhibition of PKB/Akt PH domain membrane targeting in COS-7 cells by Ins(1,4,5,6)P(4). Thus, we propose that specific inositol polyphosphates inhibit PI3-K by competing with PtdIns(3,4, 5)P(3)-binding PH domains and that this occurs mainly at the level of the downstream PI3-K target, PKB/Akt.

Polyphosphoinositides play an important role in membrane trafficking and cell signalling. In plants, two PtdInsP isomers have been described, PtdIns3P and PtdIns4P. Here we report the identification of a third, PtdIns5P. Evidence is based on the conversion of the endogenous PtdInsP pool into PtdIns(4,5)P(2) by a specific PtdIns5P 4-OH kinase, and on in vivo (32)P-labelling studies coupled to HPLC head-group analysis. In Chlamydomonas, 3-8% of the PtdInsP pool was PtdIns5P, 10-15% was PtdIns3P and the rest was PtdIns4P. In seedlings of Vicia faba and suspension-cultured tomato cells, the level of PtdIns5P was about 18%, indicating that PtdIns5P is a general plant lipid that represents a significant proportion of the PtdInsP pool. Activating phospholipase C (PLC) signalling in Chlamydomonas cells with mastoparan increased the turnover of PtdIns(4,5)P(2) at the cost of PtdIns4P, but did not affect the level of PtdIns5P. This indicates that PtdIns(4,5)P(2) is synthesized from PtdIns4P rather than from PtdIns5P during PLC signalling. However, when cells were subjected to hyperosmotic stress, PtdIns5P levels rapidly increased, suggesting a role in osmotic-stress signalling. The potential pathways of PtdIns5P formation are discussed.