Chapter 18 G-protein coupled receptors and hormone secretion

Chapter 18

G-Protein Coupled Receptors and Hormone Secretion G.J. LAW, M. RUPNIK, R. ZOREC, P.M. LLEDO, and W.T. MASON

GTP Binding Proteins: Key Molecular Switches Switching GTP Binding Proteins On and Off G Proteins: Signal Transduction and Cell Secretion Introduction to G Proteins G Protein Function Receptor-G Protein Interactions G Protein-Effector Interactions G Proteins and Cell Secretion Small GTP-Binding Proteins in Secretion: An Overall View Distribution and Function of rab Proteins Are Calcium and Small GTPases Partners Participating in Regulated Exocytosis? Heterotrimeric GTP-Binding Proteins and Exocytosis in the Adenohypophyseal Cell

Principles of Medical Biology, Volume 7B Membranes and Cell Signaling, pages 421-450. Copyright © 1997 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-812-9

421

422 422 423 423 423 424 425 426 426 428 430 432

422

G.J. LAW, M. RUPNIK, R. ZOREC, P.M. LLEDO, and W.T. MASON

Use of Synthetic Peptides to Probe the Function of GTP-Binding Proteins Receptor-G Protein Interactions Direct Effects on G Proteins Synthetic Peptides: G Proteins Role of Small GTPases in Secretion Cellular Localization of Smg's and Their Role in Transport Regulatory Proteins of Smg's Ras and Its Effector Switch as a Model for Other Smg's Synthetic Peptides: Small GTPases Rab3-Peptide Role of Rab3 in Membrane Traffic Role of ARF in Membrane Traffic Conclusion to Use of Peptides Final Comments Strategies to Assign Specific GTPases Involved in the Control of Ionic Channel and Secretory Activity Assignment of G-protein Subtypes to Specific Receptors Inducing Modulation of Ionic Channels

434 434 435 435 436 436 436 436 437 437 438 438 439 440 441 441

GTP BINDING PROTEINS: KEY MOLECULAR SWITCHES GTP binding proteins act as fundamental molecular switches controlling many important cellular responses including that of hormone and neurotransmitter release from secretory cells (Clapham, 1994; Bourne et al., 1991; Goud and McCaffety, 1991; Bomsel and Mostov, 1992; Takai et al., 1992; Conklin and Bourne, 1993; Bourne, 1988). Two different kinds of GTP binding proteins exist in cells: one being the heterotrimeric G protein involved in signal transduction across the plasma membrane; and the other, the small monomeric GTP binding proteins (smg's) involved in membrane trafficking, organization of the cytoskeleton, protein synthesis, and cellular growth (Clapham, 1994; Bourne et al., 1991; Bomsel and Mostov, 1992; Conklin and Bourne, 1993; Takai et. al., 1992; Goud and McCaffety, 1991; Bourne, 1988). Both kinds of molecular switches are only active when GTP is bound to them. In the case of heterotrimeric G proteins, this releases the active catalyst but small GTP binding proteins appear to act as cofactors in a recycling mechanism rather than as a free catalyst. Smg's appear to facilitate unidirectional transport and/or fusion of vesicles with their acceptor compartment (Bourne, 1988). Details of the role and regulation of GTP binding proteins involved in the control of secretion is a central focus of much current investigation.

SWITCHING GTP BINDING PROTEINS ON AND OFF Ligand occupied-receptors at the plasma membrane are clearly well defined as guanine nucleotide exchange factors (GNEF) which promote dissociation of GDP

G'Protein Coupled Receptors and Hormone Secretion

423

and binding of GTP to the a-subunit of the heterotrimeric G protein. This GTP binding causes the a and py subunits of the heterotrimer to dissociate to release the active catalytic subunit(s). Upon target recognition by the Ga-GTP bound catalyst its intrinsic GTPase activity may be further stimulated to accelerate the hydrolysis of bound GTP to GDP (Berstein et al., 1992); and this GTPase activity sv^itches the catalytic activity of promoting reassociation of the apy trimer. Py dimers may also play an active role in signal transduction, either alone, or in combination with activated Ga monomers (Iniguez et al., 1993). Regulation of the GDP/GTP cycle of smg's involves a separate and distinct class of GNEF's and GTPase activating factors (GAPs); but unlike G proteins their downstream targets remain largely unknown (Takai et al., 1992).

G PROTEINS: SIGNAL TRANSDUCTION AND CELL SECRETION Introduction to G Proteins

In order to communicate with the outside world cells have developed signal transduction mechanisms in which molecules bind to specific receptors on the cell-surface to activate membrane associated second messengers. These messengers translate the external stimuli into a cellular response. Hormones and neurotransmitters all work through a similar mechanism involving binding of a molecule to the extracellular surface of a transmembrane receptor that catalyzes the transfer of GTP onto a heterotrimeric G protein. There are many hundreds of different types of G protein-linked receptors. Molecular cloning of the heterotrimeric G protein shows that there are at least 21 G^, 4 Gp, and 7 G^ subunits (Clapham, 1994). The number of possible combinations of a, P, and y subunits is therefore enormous. G Protein Function

G proteins were first categorized by function, for example, G^ was shown to stimulate adenylate cyclase. Sequence homology now indicates that there are four families of G^ subunits which are listed below with some of their assigned functions. The list also includes a novel G protein which is coupled to insulin receptors (Srivastava et al., 1994) and functions for isolated py subunits (Iniguez et al., 1993). This list is not exhaustive and other functions are possible. G subunit • > «

^asl,2

Ga-oif(olfactory) ^i

^ail Gai2

Function Adenylate cyclase (+) Calcium ion channels (+) Adenylate cyclase (+) Adenylate cyclase (-) Phospholipase C (+)

424

GJ. LAW, M. RUPNIK, R. ZOREC, P.M. LLEDO, and W.T. MASON

G subunit Gai3

Go.A,B G<. ^ati 2"(transduscin) Gaq 15,16 Gcxq 11.14 G^ 12,13

G, Gu Gi,(67Kd) Gpy

Function Potassium ion channel (+) Brain-Calcium ion channel (-) Brain-PLC? Cyclic GMP phosphodiesterase (+) PLCbl(+) PLCbl(+) PTX insensitive Transforms 3T3 cells Insulin receptor-tyrosine kinase Potassium ion channels (+) PLCb2 (+) PI3-Kinase (+)

To support and identify a role for a G protein in a signal transducing pathway a number of pharmacological reagents, bacterial toxins, and venom peptides, have been used along with more specific reagents such as antibodies, antisense oligonucleotides and recombinant, or purified, G protein subunits. Reagents to Affect G-Protein Activity Reagent

Target

GTPyS

Most GTPases

GDPPS Aluminum Fluoride Pertussis Toxin Cholera Toxin Mastoparan

Most GTPases G^-subunits

Antibodies Antisense Specific Peptides and Protein Cl-ions

^ai/o

Description Slowly hydrolyzable analog which activates GTPases Deactivates many GTPases Activates G proteins No effect on Small GTPases ADP-ribosylates and blocks activation

G subunit G subunit G subunit

ADP-ribosylates and blocks hydrolysis of GTP. Permanent activation. Short-peptide mimics receptor-tail to activate G proteins. Specific inhibition Specific knockout Reconstitution of activity

G subunit

Increased/decreased hydrolysis

G„s G^ subunits

Receptor-G Protein Interactions

Heterotrimeric G proteins relay information from cell-surface receptors to effectors. These G protein-coupled receptors often share the characteristic motif of seven

G'Protein Coupled Receptors and Hormone Secretion

425

hydrophobic and transmembrane domains which are connected in series via intraand extracellular loops (Strader et al., 1989; Dohlman et al., 1991; Savarese and Fraser, 1992). Site-directed mutagenesis and chimeric receptors indicated an involvement of the intracellular loops in the interaction with G proteins (Strader et al., 1989; Dohlman et al., 1991; Savarese and Fraser, 1992). In particular, the NH2and COOH-terminal ends of the third intracellular loop have been proposed to form the major, but not the sole, sites involved in G protein coupling (Savarese and Fraser, 1992; Dohlman et al., 1991; Strader et al., 1989). Deletions or substitutions in these terminal ends of the |32 adrenergic receptor caused a marked dimunition of agonistinduced adenylate cyclase activity. In contrast, replacement of residues of key amino acids in the C terminal end of the third intracellular loop of the a-adrenergic receptor produced a receptor that displayed a 100-fold higher affinity for its ligand (noradrenaline) and a 300-fold increase in the potency for activating PI breakdown (Cotecchia et al., 1990). This remarkable data demonstrates that changes in the amino acid sequence of a putative effector loop can dramatically improve the ability of a transmembrane receptor to couple to a G protein. Charged amino acid residues appear to frequent effector loop regions involved in coupling to G proteins; but secondary structure may also be important. Secondary structure predictions of terminal ends of the third intracellular loop indicated that they form amphipathic a-helices; and this a-helical structure was proposed as the main determinant in the interaction with G proteins (Strader et al., 1989). Data suggest that a-helical distorting substitutions disrupt coupling between m3 muscarinic receptors and G proteins in a Xenopus laevis oocyte expression system (Duerson et al., 1993). Compelling evidence also shows that the extreme carboxy-terminus of the receptor directs contact to Ga s polypeptide at least in the S49 unc mutant cells. In this cell-line, a point mutation of Arg389 (wild type) to Pro (unc mutant) was found to uncouple the ligand-occupied receptor from coupling to Gs (Sullivan et al., 1987). This effect is thought to be a consequence of disruption of the C-terminal a-helix by proline. Recent work has shown that the py subunits may also play a crucial role in the determination of which G protein interacts with which transmembrane receptor (Kleuss et al., 1993a; Kleuss et al., 1993b). G Protein-Effector Interactions

It is beyond the scope of this chapter to say much about how G proteins couple to their downstream effectors (but for a recent review on this subject see Conklin and Bourne, 1993). At least some of these effectors (e.g., PLC), however, have been shown to stimulate GTPase activity to regulate G protein activity (Berstein et al., 1992). Another remarkable finding has been that substitution of three amino acids in one G protein can switch its downstream specificity to that of another (Conklin etal., 1993).

426

G.J. LAW, M. RUPNIK, R. ZOREC, P.M. LLEDO, and W.T. MASON

G Proteins and Cell Secretion

Most heterotrimeric G proteins linked to cell-surface receptors can regulate secretory output via their effects on membrane associated second messengers. Regulation of secretory output is often associated with G protein activation of the phospholipase C (PLC) signaling cascade. This leads to an increase in intracellular Ca^^ ion concentration due to production of water soluble inositol 1,4,5-trisphosphate (IP3), and activation of protein kinase C via the increase in diacylglycerol (DAG). Other protein kinases (e.g., cAMP and Ca^VCalmodulin) and ion channels may also play an important role in regulation of secretion. A key finding has been the remarkable effect of GTPyS to stimulate secretion in metabolically poisoned and permeabilized cells even in the presence of inhibitors of PLC activation (Tatham and Gomperts, 1991). This has been interpreted to suggest a role for a late action of a GTP binding protein in the control of exocytosis (Tatham and Gomperts, 1991). Antibodies to specific G protein have been reported to block effects of GTPyS on release (Aridor et al., 1993; Vitale et al., 1993). The role of G proteins in the control of exocytosis may be to have a direct effect on the formation of a fusion pore which is analogous to their proposed role on gating of ion channels in the plasma membrane (Clapham, 1994). Heterotrimeric G proteins have been detected in many intracellular compartments of the secretory pathway in addition to the plasma membrane (Bomsel and Motsov, 1992). Evidence is beginning to accumulate that a pertussis toxin-sensitive G^ -3 localized to the Golgi is involved in the control of the rate of constitutive secretory protein transport through this compartment in normal cells (Stow et al., 1991). Multiple trimeric G proteins now appear to exist on the trans-Golgi network and exert stimulatory and inhibitory effects on secretory vesicle formation (Leyte et al., 1992). Regulation of these G proteins is unknown. Small GTP-Binding Proteins in Secretion: An Overall View

GTPases belong to a large superfamily of conserved proteins built according to a common structural design and sharing a molecular mechanism. Each protein in the family is a precisely engineered molecular switch that can change its affinities for other macromolecule complexes. We refer here to members of this superfamily as "GTPases" rather than "GTP-binding proteins" to evoke the crucial biochemical activity of these proteins which involves a molecular cycle rather than just a binding process. Hence, turn "on" by binding guanosine triphosphate (GTP) and "off' by hydrolyzing GTP to guanosine diphosphate (GDP), the switch mechanism is remarkably versatile, enabling different GTPases to fulfill a wide range of regulatory functions in all organisms (Figure 1). Superimposed on the central concept of the two interconvertible conformational states (GDP or GTP-bound form), evolution has modified and expanded the range of input and output signals to produce a diverse set of regulatory pathways. The

G-Protein Coupled Receptors and Hormone Secretion GTP

427

GDP

GDI ^ G D P p

^ y r y p —

•=—-•"

(Guanine nucleotide dissociation inhibitor)

GAP p.

(GTPase activating protein)

Figure T. Regulation of ras-like GTPases activity. The guanine nucleotide release factor (GRF) turns "on" the GTPase by catalyzing exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). The GTPase-activating protein (GAP) catalyzes conversion of GTP-bound form back to the GDP state and therefore turns "off the protein. The guanine nucleotide dissociation inhibitor (GDI) affects nucleotide dissociation and GAP attack, but is thought to have also other major roles such as membrane localization or protein solubility.

best understood of the GTPases are those controlling peptide elongation in Escherichia coli (elongation factor or EF-Tu) and signal transduction across the plasma membrane in manmialian cells (heterotrimeric G proteins). However, a large distinct group of GTPases has emerged somewhat unexpectedly out of oncogene research. This family comprises the ras gene products which were first highlighted in the early 1980s as sites of somatic mutation in human cancers, and since then a great deal of effort has been put into understanding their function. Since this family of genes encodes a novel class of regulatory GTPases involved in the control of cell proliferation, differentiation, and vesicular transport, we will focus our presentation on the cellular functions of these proteins structurally related to the ras oncoprotein. This superfamily includes the ras, ral, rac, rho, rap, and rab gene families in mammalian cells. It currently comprises over 50 members, which have been indeed found to regulate a large spectrum of elementary cellular processes. All members of the ras superfamily share at least 30% homology to ras protein. This homology principally involves some conserved sequence motifs that comprise the guanine nucleotide-binding domain and are critical for GDP/GTP binding, GDP exchange and GTP hydrolysis. A second domain that is strongly conserved among members

428

G.J. LAW, M. RUPNIK, R. ZOREC, P.M. LLEDO, and W.T. MASON

of a given gene family is the effector domain. The effector domain in ras is essential for interaction with a GTPase activating protein (GAP), a protein that stimulates the low intrinsic GTP-hydrolysis rate of cellular ras to maintain it in the GDP-bound form ("off') state. This domain is also generally conserved within each family. Most small GTPases possess, however, some hypervariable regions such as the carboxyl terminus that contains a CAAX, CXC, or CC motif that is essential for membrane association and which is modified post-translationally by proteolytic processing, polyisoprenylation, carboxymethylation and, in some cases, palmitoylation. High-resolution structures of both the GDP- and GTP-bound forms of Ras protein have been solved. Analysis of these structures reveals that the two regions, the effector domain and the region encompassing residues 63-70, undergo considerable conformational change depending on whether GDP or GTP is bound. These "switch" regions are therefore likely to play an essential role in the recognition of upstream or downstream effector proteins. The potential role of ra^-related small GTPases in regulating vesicular transport was first realized in yeast when a SEC4 gene product (SEC4) was found to control the constitutive secretory pathway. The sequence of SEC4 indicated that it is a Ras-like protein and subsequent analyses revealed that it is present on the surface of secretory vesicles and can bind and hydrolyze GTP. A search for the mammalian counterparts of SEC4 led to the identification of a large number of ras-related GTPases that may include more than 30 different gene products. They have been grouped into a family termed Rab proteins for 'Ras-like proteins from rat brain.' They have in common not only their structural features, but also their ability to regulate the signaling pathways regulating vesicle traffic and sorting at the plasma membrane. By analogy with other guanine nucleotide-binding proteins, the Rab proteins are considered inactive in their GDP-bound form. This form is stabilized by guanine nucleotide dissociation inhibitors (GDI). Upon stimulation by a guanine nucleotide releasing protein (GNRP), the GDP is exchanged for GTP, and the Rab proteins switch to their active, GTP-bound form. This state is quasi-irreversible until the rab proteins hydrolyze the bound GTP to GDP, this effect being stimulated by a GAP. Thus, the proteins become inactivated, allowing the process to be repeated. Hence, as with other GTPases, Rab proteins behave as molecular switches cycling from an active (GTP-bound) to an inactive (GDP-bound) conformation. Since the general role of GTPases is to control the specificity and the temporal coherence of intermolecular recognition processes, it is very likely that Rab proteins act as regulators of vesicular targeting from an upstream to a downstream compartment (Figure 2). Distribution and Function of Rab Proteins

Several GTPases have now been implicated directly or indirectly in regulation of vesicular transport through the secretory pathway of eukaryotic cells. In yeast, these

G'Protein Coupled Receptors and Hormone Secretion

429

Donor Membrane

T u

Carrier Vesicle

II

RAB

GAP GDI

\^m GEF 1

Acceptor Membrane Figure 2. Model for a possible functional mechanism of Rab proteins. In step 1, vesicles budding induces the formation of a carrier vesicle from a donor membrane. This vesicle is associated v^ith the active, GTP-bound form of Rab protein that controls the vesicular traffic within the cell. The interaction of the vesicle with the correct acceptor membrane induces GTP hydrolysis catalyzed by GAP (step 3). The resulting GDP-bound form (step 2) of the Rab protein, in step 4, is solubilized by GDI. Then, the Rab protein is recruited from its soluble form by interaction with a GEF that is associated with the donor membrane (step 5). By triggering dissociation of the bound GDP, the GEF causes the Rab to be released from the GDI and thus facilitates membrane attachment.

proteins include YPTl, ARF, and SARI, which are required for endoplasmic reticulum to Golgi, or intra-Golgi transport, and SEC4, v^hich is required for delivery of secretory vesicles to the cell surface. Genetic mutants of yeast deficient for the function of SEC4 and YPTl have provided the strongest evidence that these proteins may control the vectorial traffic of intracellular vesicles. Immunolabelling studies shov^ YPTl associated w^ith the Golgi stack and SEC4 is present on the membrane vesicles. Like ras, SEC4 is synthesized as a soluble precursor that rapidly associates with the plasma membrane and transport vesicles, is predominately found in the GDP-bound form, and has a low intrinsic rate of hydrolysis. Since transport vesicles accumulate in SEC4 mutants, it is clear that SEC4 is not required for vesicle formation, rather, it is required for vesicle targeting and/or fusion.

430

G.J. LAW, M. RUPNIK, R. ZOREC, P.M. LLEDO, and W.T. MASON

The mammalian protein Rabl is the functional homolog of YPTl since it has been shown that Rabl protein can substitute for YPTl function in yeast. Similarly, Rab3 is a functional homolog of SEC4, since it has been reported that Rab3A is specifically expressed in synaptic vesicles and dense core granules in neurons and neuroendocrine cells where it could control the recruitment of synaptic vesicles and their fusion with the plasma membrane during exocytosis. Other members of the Rab3 subfamily, termed RabSB and RabSC, have also been found associated with the membrane of secretory vesicles or dense core granules of neurones, endocrine and neuroendocrine tissues. Rab3D is predominantly expressed in adipocytes and mast cells. Adipocytes possess a pool of vesicles containing a glucose transporter (GLUT4). Upon stimulation with insulin, these vesicles undergo exocytosis, and GLUT4 is integrated into the plasma membrane in order to enhance the capacity for glucose uptake. It is now clear that each Rab family member is distributed throughout the exocytotic and endocytotic pathways with distinctive subcellular distributions within cells, and most of the organelles involved in exocytosis or endocytosis possess at least one distinct member of this family. Depending essentially upon the sequence of their carboxy-terminus, each member of the Rab protein family seems to be associated with a particular vesicular compartment. In addition, a post-translational isoprenylation of cysteine residues at the carboxy-terminus of the Rab proteins is required for their functional association with the vesicular membrane. Although most of the Rab family members are uniformly distributed in mammalian cells, controlling the regulated exocytosis, they could also be involved in more differentiated secretory or compartmentalized processes specific to neurons, endocrine, or epithelial cells. Are Calcium and the Small GTPases Partners Participating in Regulated Exocytosis?

An intriguing set of observations has led to a possible link between the function of small GTPases and calcium in regulation of vesicular transport. Indirect evidence for the role of calcium in vesicular transport was provided by the ability of elevated extracellular calcium concentrations to reverse the transport defect imposed by growth of a temperature-sensitive YPTl mutant at the restrictive temperature. This result led to the speculation that YPTl regulates vesicle transport in a concerted manner with the cytosolic calcium concentration. In the mammalian transport assay, optimal transport between the ER and the Golgi, and between Golgi stacks is observed with free calcium concentrations of 50 to 500 nM, suggesting that the normal physiological oscillations of free calcium concentration observed in vivo are adequate to support maximal vesicular traffic. It has long been known that excitable tissues release their chemical signals, neurotransmitters or hormones, via a process that is regulated by calcium ions. One candidate for the calcium-sensitive regulator is the synaptic vesicle protein synap-

G-Protein Coupled Receptors and Hormone Secretion

431

Miinc-1 i

Synaptophysin

Rab3 GDP

Plasma Membrane

Syntaxins Neurexins Ca2+ channel

Fusion pore

Figure 3. Schematic representation of proteins associated either with secretory vesicles or the plasma membrane at the nerve terminal. This diagram depicts characterized synaptic vesicle proteins and some of their postulated receptors and functions. The molecular machine for docking the vesicle have been represented as distinct from the molecular fusion machinery, for more clarity. Synapsins are vesicle-associated proteins that are thought to mediate interactions between the synaptic vesicle and the cytoskeletal elements of the nerve terminal. The docking fusion, and release of vesicles appear to involve several distinct interactions between vesicle proteins and proteins of the nerve terminal plasma membrane. These proteins include synaptotagmins, the VAMP (also called synaptobrevin) and rabphilin on the vesicle membrane and syntaxins, SNAP-25 and neurexins on the nerve terminal membrane. In neurones, it can be proposed that synaptotagmin and rabphilin might act in concert to control neurotransmitter release by sensoring calcium rises into the cytosol. Rab3 proteins control vesicle trafficking within the cell and may regulate the calcium-sensitivity of the fusion machinery involved in exocytosis. NSF/SNAPs constitutes the fusion machinery.

totagmin which is composed of an amino-terminal intravesicular domain, and a cytoplasmic domain made up of two repeats homologous to the C2 regulatory domain of protein kinase C. These C2-homologous domains have been implicated in the calcium-dependent interaction of synaptotagmin with membrane phospholipids. Thus, it has been proposed that synaptotagmin acts as a calcium sensor on the surface of the synaptic vesicle. Indeed, the interaction of synaptotagmin with a presynaptic plasma membrane protein, syntaxin, coupled with the interaction of syntaxin with voltage-gated calcium channels, place synaptotagmin in an ideal position to respond to calcium. In this context, it is interesting to note that rabphilin,

432

G.J. LAW, M. RUPNIK, R. ZOREC, P.M. LLEDO, and W.T. MASON

one of the interacting partners of GTP-bound Rab3 protein, also shows sequence homologies to the C2 domains of synaptotagmin. The C2 domains of rabphilin provides a link between Rab3 protein found on the membrane of secretory vesicles and calcium signaling. Therefore, it can be proposed that synaptotagmin and rabphilin might act in concert to control neurotransmitter release and that Rab protein will regulate the calcium-sensitivity of the fusion machinery (including synaptotagmin and rabphilin) involved in exocytosis (Figure 3). Through this mechanism, Rab3 proteins might be responsible for desensitization processes which occur at the presynaptic terminals and might be associated with adaptive processes involved in higher brain function such as, for example, long-term potentiation or memory. Heterotrimeric GIF-Binding Proteins and Exocytosis in the Adenohypophyseal Cell

GTP-binding proteins link receptors with a number of effectors in cellular signaling pathways. Downstream to the signaling pathways, an additional GTPbinding protein (G^) was proposed to regulate exocytotic fusion (Gomperts, 1990). Despite the fact that, in the last years the identification of this protein has received considerable attention, we still have not reached the final answer. The rest of this section is to discuss our present knowledge about the role of GTP-binding proteins in exocytosis of the adenohypophyseal cell. Sikdar et al. (1989) have shown that GTP-binding proteins are involved in the regulation of secretory activity of single adenohypophyseal cells. In the presence of nonhydrolyzable GTP analogs, Sikdar et al. (1989) have observed two effects, early and delayed, on calcium induced secretory activity monitored by measurements of membrane capacitance, proportional to surface area (Neher and Marty, 1982). The early, stimulatory effect was evidenced by the augmented rate in membrane capacitance increase, while the delayed inhibitory effect was manifested as a reduction in the steady state increase in membrane capacitance. The inhibitory effect in the presence of nonhydrolyzable GTP analogues in pituitary cells may be due to attenuated delivery (translocation and docking) of secretory organelles to the plasmalemma (Sikdar et al., 1989), and was also observed in neurones (Hess et al., 1993). The two groups of GTP-binding proteins: heterotrimeric and monomeric (Bourne et al., 1990) were suggested as candidates for a role in the control of exocytotic activity in secretory cells. The control of trafficking and vectorial transport of vesicles in the secretory pathway is attributed to monomeric GTP-ases of the Ras superfamily. Rab3 members of this family are believed to play a role in regulated exocytosis (Pfeffer, 1992; Simmons and Zerial, 1993). An indication for a role of these proteins in exocytosis comes from experiments where putative effector domain peptides of Rab3 were introduced into mast cells (Oberhauser et al., 1992), chromaffin cells (Senyshyn et al., 1992), pancreatic cells (Padfield et al., 1992) and

G-Protein Coupled Receptors and Hormone Secretion

insulin secreting cells (Li et al., 1993), which induced exocytosis in the absence of calcium. These peptides stimulated exocytotic fusion in an in vitro system also (Edwardson et al., 1993). On the other hand, putative effector domain peptides of Rab3a had no effect (Okano et al., 1993; Morgan and Burgoyne, 1993), or were even found to inhibit exocytosis (Davidson et al., 1993). Furthermore, the most effective effector domain Rab3a peptide carried mutations, which in full protein, abolished the interaction with all known target proteins and regulatory elements (McKieman et al., 1993). Moreover, a mastoparan like effect of these peptides was described as well (Law et al., 1993). Henceforth, the effector domain Rab3a peptide studies should be considered critically. More direct evidence for a role of Rab3 in regulated exocytosis comes from experiments where the expression of Rab3 protein was inhibited by antisense probes. Using this molecular biology approach combined with patch-clamp recording, it was shown that Rab3b proteins are required for Ca^^-induced secretory activity of adenohypophyseal cells (Lledo et al., 1993). In adrenal medulla chromaffin cells, the introduction of recombinant Rab3a proteins revealed an inhibitory role of these proteins in exocytosis (Johannes et al., 1994). Heterotrimeric GTP-binding proteins may also play a role in exocytosis (Lillie et al., 1992). However, the characterization of these GTP-binding proteins may prove to be difficult, since there are more than 16 different a subunits of heterotrimeric GTP-binding proteins (Bourne et al., 1990). In addition, as was found for the GTP-binding proteins involved in the control of voltage gated Ca^"^ channels, different Py subunits bound to a subunits may determine the functional role of the heterotrimeric GTP-binding proteins (Kleuss et al., 1992). Aridor et al. (1993) have analyzed the pertussis toxin-sensitive (PTx) heterotrimeric GTP-binding proteins in mast cells, as potential G^ candidates, since pertussis toxin pretreatment of these cells inhibits exocytosis. They found an activation of exocytosis by the heterotrimeric GTP-binding protein G-3. To see whether the secretory activity of a single adenohypophyseal cell is sensitive to PTx, we pretreated rat melanotrophs with PTx (250 ng/ml, 7 hours) and recorded secretory responses by monitoring changes in membrane capacitance using the patch clamp technique (Zorec et al., 1991). As shown on Figure 4, in PTx pretreated cells Ca^'^-insensitive secretory activity was recorded, suggesting that a step downstream of other calcium dependent steps requires a PTx-sensitive heterotrimeric GTP-binding protein for vesicle fusion to take place. This activity was blocked by GDPpS (Rupnik and Zorec, 1994), which indicates that the PTx treatment might lock the heterotrimeric GTP-binding protein in the GTP bound state. In contrast to nonexcitable cells (Aridor et al., 1993), PTx pretreatment of excitable melanotrophs resulted in increased secretory activity. This may indicate that a PTx sensitive heterotrimeric GTP-binding protein is coupled to the exocytotic control pathway of melanotrophs in a stimulatory sense, whereas in mast cells it may have an inhibitory role.

433

434

G.J. LAW, M. RUPNIK, R. ZOREQ P.M. LLEDO, and W.T. MASON

Ca' -free + PTX

1 pF

H

200 s Figure 4. Patch clamp recordings in a secretory ceil. Representative changes in Cm recorded in a control (bottom) and a pertussis toxin pretreated cell (250 ng/ml, 7 hours) {top). Pipette and bath solutions as in Rupnik and Zorec (1994). The noncompensated method of membrane capacitance measurements was used (Lindau and Neher, 1988; Zorec etal., 1991).

Use of Synthetic Peptides to Probe the Function of GTP-Binding Proteins The aim of this section is to discuss how synthetic peptides to different regions of transmembrane receptors and GTP binding proteins have increased our understanding of the molecular mechanism of signal transduction and signaling pathways involved in the control of membrane traffic and fusion in secretory cells. Receptor-G Protein Interactions One possible limitation of data obtained from site-directed mutagenesis is that this may produce unfavorable pertubations of the protein-folding and structure. To corroborate these findings, studies have been carried out using synthetic peptides derived from various regions of the receptor. Synthetic peptides of the second, third and fourth intracellular loop of the P-adrenergic receptor have been shown to compete synergistically with the hormone-stimulated receptor for G^ activation pointing to the involvement of multisite contacts (Munch et al., 1991). A highly charged peptide sequence (KASRWRGRQNREKRFTF) corresponding to the Cterminal end of the third intracellular loop of a 2aR-receptor is sufficient alone to activate GTP binding to G^- (Ikezu et al., 1992). A similar finding was previously reported for a charged peptide sequence (RVGLVRGEKARKGK) isolated from the IGF11/M6P receptor (Okamoto et al., 1990). Recent studies with synthetic peptides derived from the dopamine D2 receptor refute the idea that an amphipathic a-helical structure predicts the ability of a peptide to interact with a G protein (Voss et al., 1993). Peptide D2N (VYIKIYIVLRRRRKRVNTK) was the only sequence tested that had a specific effect on D2 coupling and was shown to stimulate a pertussis toxin-sensitive G protein. D2N did not exhibit a predicted a-helix, whereas other regions which did had no effect (Voss et al., 1993). A remarkable

G'Protein Coupled Receptors and Hormone Secretion

435

feature of D2N is its cluster of positive charges (...RRRRKR...) which are also necessary for activity of other polybasic peptides. Direct Effects on G Proteins

Mastoparan is a polybasic peptide (INLKALAALAKKIL) which was isolated from wasp venom and was found to stimulate secretion of histamine from intact mast cells (Hirai et al., 1979). The finding that PTx inhibited mastoparan-stimulated histamine secretion indicated that a G protein might mediate this effect of mastoparan on mast cells (Higashijima et al., 1988). PTx specifically catalyzes the ADP-ribosylation on a cysteine residue close to the carboxy terminus of the a-subunit of several G proteins and renders them insensitive to regulation by the receptor, although their ability to hydrolyze GTP and to stimulate effector proteins still remains intact (Van Dop et al., 1984). Further work showed that mastoparan binds to G proteins and promotes GDP/GTP exchange via a mechanism that is strikingly similar to that of ligand occupied-receptors (Higashijima et al., 1990; Weingarten et al., 1990). Mastoparan also interacts with smg's, rho and rac, to inhibit ADP-ribosylation of these proteins by Clostridium botulinum exoenzyme C3 but further details are lacking (Koch et al., 1991). Mastoparan forms an amphipathic a helix when it binds to phospholipid membranes and presents three positive charges to the aqueous face (Wakamatsu et al., 1992). Sites important for interaction of receptors with G proteins are also positively charged as exemplified by the case of D2N described above. Nine peptides representing sequence permutations of mastoparan were tested to investigate the rules governing the interaction with G proteins (Oppi et al., 1992). Their specificity was remarkable and when compared to the authentic sequence of mastoparan effects ranged from super-active analogs, inactive ones and sequences that inhibited basal GTPase activity of G^ ^. An encouraging note was that one of the active mastoparans had a high sequence homology to human neurofibromatosis related protein (NFl, a putative GTPase activating protein). Comparison of the amino acid sequence of other venom peptides coupled to activation of G proteins indicates that a cationic cluster at one side of the a-helical surface was more important for activity than a specific, a-helical structure (Tomita et al., 1991), and this observation is consistent with others (Oppi et al., 1992; Voss et al., 1993). At present, however, there is no hard data to predict what peptide sequences are likely to interact with a selected GTP binding protein beyond those obtained from empirical observations, such as clusters of positive charges and perhaps the propensity to form an amphipathic a-helix for correct charge distribution in some cases. Synthetic Peptides: G Proteins

A powerful approach has been to use both peptides and antibodies to different G proteins to define which ones are important in signal transduction and release. Carboxy-terminal peptides to G proteins have been shown to prevent coupling of

436

G.J. LAW, M. RUPNIK, R. ZOREC, P.M. LLEDO, and W.T. MASON

G proteins to their respective receptors (Hamm et al., 1988; Aridor et al., 1993). In particular, residues 345-354 of G^.3 (KNNLKECGLY) were found to specifically block a stimulation of release from mast cells (Aridor et al., 1993). In addition to this, an antibody to G(^-3 was found to cause a specific inhibition of GTPyS induced release in permeabilized mast cells (Aridor et al., 1993). These probes to G proteins should be easy to use to provide more interesting data in the future.

ROLE OF SMALL GTPASES IN SECRETION Cellular Localization of Smg's and Their Role in Transport

Evidence for a role of smg's in vesicular transport and fusion with the plasma membrane came from studies with yeast secretory mutants (Salminen and Novick, 1987). In the absence of one of their smg's identified as the Sec4 gene product secretory vesicles were found to accumulate and failed to fuse with the plasma membrane (Goud et al., 1988). Identification and cellular localization of other smg's throughout the secretory pathway in mammalian cells switched attention to the rab and arfgcnt family (Goud and McCaffety, 1991). Understanding how smg's might act to facilitate the efficient unidirectional transport of membrane traffic from a donor to an acceptor compartment has become a major focus of research into protein trafficking in cells. Regulatory Proteins of Smg's

Smg's act as regulatory switches whose activity is controlled by cycling between the active GTP bound and inactive GDP states. Unlike G proteins, smg's have slow intrinsic rates of activation and inactivation and the hunt for regulatory proteins for both arms of the cycle began. Two classes of regulatory proteins exist: the guanine nucleotide exchange factors (GNEFs), which catalyze release of bound GDP, promoting its replacement by GTP, and the GTPase-activating proteins (GAPs), which speed up GTP hydrolysis as an irreversible step in the cycle. GNEFs can be either stimulatory or inhibitory (for a recent review see Takai et al., 1992). Ras and Its Effector Switch as a Model for Other Smg's

Ras p21 is an smg known to play a pivotal role in regulating growth and differentiation in most eukaryotic cells (Barbacid, 1987). Site-directed mutagenesis studies of ras identified its effector domain to amino acid residues 32-40 (Sigal et al., 1986). Comparison of the crystal structure of the GTP-bound, "on switch," and GDP-bound, "off switch," revealed considerable conformational change in this "effector" region (Milbum et al., 1990). This effector region is commonly referred to as "switch 1" of the "L2/p2-loop" of ras which forms an exposed molecular surface available for possible interaction with an unknown downstream target. A switch 2 region (key residue Y64) is also important for GAP activation of ras.

G'Protein Coupled Receptors and Hormone Secretion

437

Specific details are beyond the scope of this chapter and so the reader is referred to two recent minireviews for more information (Polakis and Cormack, 1993; Stouten etal., 1993). The crystal structure of the a-subunit of the G protein, transducin, has recently been described (Noel et al., 1993). There is a remarkable similarity to the framework used to model ras p21 structure to function. G^^-subunits appear to differ from small GTPases by having an additional domain inserted into a site corresponding to loop 2 of ras p21. This extra domain acts like a teapot lid to restrict movement in and out of the guanine-nucleotide binding pocket of the core domain. Receptors must somehow open this lid for activation. In any event, the core-domain of G proteins and small GTPases appears to be remarkably similar in structure even though the percentage similarity in identity of their primary structure is low. Synthetic Peptides: Small GTPases

Short-peptides corresponding to amino acid residues of the "switch 1" effectorregion of Ras were found to be effective in Ras-GAP protein interactions (Schaber et al., 1989). By analogy and appropriate alignment of protein sequences effector domain peptides to other smg's were defined and tested. The case for peptides to Rab3 and Arf are discussed below. Rab3-Peptide Authentic peptide sequence of a putative effector domain of Rab3 protein (VSTVGIDFKVKTIYRN) was first shown to partially inhibit ER to Golgi transport (Plutner et al., 1990). Site-directed mutagenesis studies had previously shown that substitution of the two amino acids TV with AL inhibited the ability of GAP to stimulate Ras-GTPase activity in vitro; and this modified peptide sequence (VSALGIDFKVKTIYRN) known as Rab3AL was found to completely inhibit transport (Plutner et al., 1990). Localization and dissociation of Rab3a protein from synaptic vesicles during exocytosis (Fischer von MoUard et al., 1991) may well have encouraged a number of workers to look for effects of Rab3-like peptides in regulated exocytosis. Since then, Rab3-like peptides have been shown to stimulate release from pancreatic acinar cells (Padfield et al., 1992), chromaffin cells (Senyshyn et al., 1992); and perhaps most convincingly to degranulate mast cells (Oberhauser et al., 1992; Law et al., 1993). Rab3-like peptides also appear to stimulate membrane fusion between isolated secretory granules and plasma membranes (Edwardson et al., 1993). Effects of Rab3AL and the authentic effector domain sequence appeared to be specific because synthetic peptides made to different regions of Rab3, and to aligned sequences of other smg's were without effect (Law etal., 1993). Interestingly, Rab3AL was shown to have a similar effect to that of other polybasic peptides such as mastoparan which lowers the intracellular cAMP concentration via a pertussis-toxin sensitive G protein (Law et al., 1993). At present

438

G.J. LAW, M. RUPNIK, R. ZOREQ P.M. LLEDO, and W.T. MASON

there is no further evidence to suggest that Rab3 protein works via a PTX-sensitive G protein. Some caution might then be required to assume that all the effects of this peptide are due to Rab3 protein. One can speculate that specific docking of the Rab3 effector domain to a PTX-sensitive G protein might play a role both in membrane recognition and kinetic proofreading of this event. Role of Rab3 in Membrane Traffic

Taking a different approach antisense-oligonucleotides have been used to reduce expression of Rab3 proteins and this work provided evidence to support a role of Rab3b, but not 3a, in the control of regulated exocytosis in pituitary cells at the level of granule supply rather than fusion (Lledo et al., 1993). More recent work with recombinant Rab3 A protein has shown an inhibition of secretory output from chromaffin cells (Holz et al., 1994; Johannes et al., 1994). Details about the mechanism of action of Rab3 proteins remain to be elucidated. The most attractive Rab3 effector molecule so far is rabphilin-3a which contains Ca^"*" binding motifs and is similar to synaptotagmin another protein associated with synaptic vesicles (Shirataki et al., 1993). More work is required to clearly delineate the signaltransducing role of Rab3 proteins in the control of vesicle progress through the secretory pathway. For a recent review on this subject see (Fischer von Mollard et al., 1994). Role of ARF in Membrane Traffic

Recent evidence has implicated a role for a small monomeric GTP binding protein known as ADP-ribosylation factor (ARF) in the control of a wide range of vesicle transport and fusion steps along the secretory pathway using either cell-fractions and/or permeabilized cells (Kahn et al., 1993). This may include budding, transport and fusion steps required to control membrane dynamics of the nuclear-envelope (Bowman et al., 1992), endoplasmic-reticulum (Balch et al., 1992), Golgi (Kahn et al., 1992; Donaldson et al., 1992), secretory vesicle (Morgan and Burgoyne, 1993), endosome (Lenhard et al., 1992), and plasma membrane (Regazzi et al., 1991). ARF was originally identified as a protein cofactor required for efficient ADPribosylation of the trimeric G^ protein by cholera toxin (Eckstein et al., 1979). Further work suggested that ARF somehow participates in the regulation of assembly and dissembly of coat proteins on membranes which might be necessary to direct the vectorial transport of vesicles between two different compartments of the cell (Kahn et al., 1992). Recent work has now shown that ARF is necessary to stimulate phospholipase D (PLD) activity (Brown et al., 1993; Cockroft et al., 1994). These alterations in lipid content by PLD may indeed be important determinants in the control of regulated exocytosis (Stutchfield and Cockroft, 1993).

G'Protein Coupled Receptors and Hormone Secretion

439

Arf-peptides

N-terminal ARF P13 peptide (GNIFANLFKGLFGKKE) has proven to be a potent inhibitor of activity which can be stimulated by the addition of recombinant ARF protein (Balch et al., 1992; Kahn et al., 1992; Lenhard et al., 1992). It also inhibits a wide range of other activities which have been proposed to employ ARF protein inside the cell (Balch et al., 1992; Kahn et al., 1992; Donaldson et al., 1992; Lenhard et al., 1992; Morgan and Burgoyne, 1993). This includes secretion from digitonin permeabilized chromaffin cells (Morgan and Burgoyne, 1993). ARF-P13 peptide readily forms an amphipathic a-helix when exposed to a hydrophobic environment and physically resembles mastoparan (Kahn et al., 1992). Although ARF-peptide has the potential to be a GNEF acting like mastoparan, this activity would appear to oppose actions of ARF protein on Golgi-transport (Kahn et al., 1992). It remains a possibiUty that the mastoparan-like effects of ARF-peptide participate in the effect of ARF to modulate sensitivity of G^ to cholera toxin (Kahn etal., 1992). ARF-P14 peptide (IPTIGFNVETVQYKNI) corresponds to the aligned sequence in ARF to the effector domain of p21 ras (Balch et al., 1992). Although this peptide was originally reported to have no effect on ARF function (Balch et al., 1992; Kahn et al., 1992), recent work has shown that it inhibits release in patch-clamped melanotrophs as did brefeldin A (Rupnik et al., unpublished data). Recent work has also focused on expression of dominant inhibitory mutants of ARF protein in cells to substantiate the role of ARF in the regulation of membrane traffic in the ER to Golgi (Dascher and Balch, 1994; Zhang et al., 1994). Work with ARF protein on regulated exocytosis remains to be done. Brefeldin A

Other evidence to support a role for ARF in control of secretion has come from the inhibitory effect of brefeldin A. Brefeldin A has been shown to inhibit guanine nucleotide exchange on ARF, and this effect is expected to rundown activity of ARF protein inside cells. Brefeldin A inhibits protein transport from the Golgi to the cell-surface (Zeuzem et al., 1991; Miller et al., 1992). Conclusion to Use of Peptides

Synthetic peptides derived from endogenous protein sequences are clearly powerful and easy tools to make for probing receptor coupling and activity of G proteins, and other proteins for that matter. Peptides can be viewed to act as small molecules with site-specific actions. They have considerable potential to provide insight into protein-protein interactions inside the cell. Peptides with strong amphipathic and cationic properties need to be treated with some caution as they are likely to have multiple effects in cells on a diverse range of targets including GTP binding proteins, calmodulin (Fisher et al., 1994) and protein kinases (Aderem, 1992). At

440

G.J. LAW, M. RUPNIK, R. ZOREQ P.M. LLEDO, and W.T. MASON

high concentrations non-specific disruptions of Hpid bilayers are also possible. Data obtained with synthetic peptides alone is not sufficient to prove an activity is present in the full-length protein. Leads coming from work with synthetic peptides need to be confirmed with data obtained from using other pharmacological probes, bacterial toxins, antibodies, antisense-oligonucleotides, and purified protein. Final Comments

The role GTP binding proteins in signal transduction and regulation of the secretion is a subject of intense debate. Much is known about the identity and presence of GTP binding proteins in cells and about their possible effects on secretion, but specific details on signal-transduction remain to be established. We need to identify effector target protein/lipid which are necessary for each of these GTPases to work and to understand how this signal can be integrated into a secretory response. In the case of regulated exocytosis, we already know that soluble factors lost from permeabilized cells are required to retain a secretory response to Ca^"^ ions and GTPyS. This finding has encouraged work to screen for soluble proteins to reconstitute this loss of activity (Burgoyne, 1993). A strong candidate to test here might be the cytosolic ARF protein as well as others. Other clues as to which proteins may play a key role in targeted membrane fusion have also recently come from studies with botulinum-neurotoxins. These toxins appear to act as a set of rather specific and site-directed Zn^"*" proteases which can cleave and inactivate individual target proteins associated with synaptic vesicles and block release (Montecucco and Schiavo, 1993). Regulated release of synaptic vesicles provides a unique model to study rapid biochemical changes and membrane fusion which must occur over the short time scale of a fraction of a second. In contrast to this, the rate-limiting step controlling the release of dense-core granules could well be different as this often takes many seconds to achieve. Clearly much more work is required to elucidate one coherent and complete signaling pathway to explain how an exocytotic event may be triggered.

STRATEGIES TO ASSIGN SPECIFIC GTPASES INVOLVED IN THE CONTROL OF IONIC CHANNEL AND SECRETORY ACTIVITY Assignment of G-protein Subtypes to Specific Receptors Inducing Modulation of Ionic Channels

Although heterotrimeric G-proteins were initially thought to be primarily involved in the regulation of second messenger systems, it has become evident that they also are intimately involved in the regulation of ion channels either directiy or indirectly. Membrane receptors which possess seven transmembrane-segments are believed to

G'Protein Coupled Receptors and Hormone Secretion

441

activate heterotrimeric G-proteins in order to achieve their cellular effects. Therefore, G-protein activation is likely to be the first step in receptor-elicited ion channel modulation. G-proteins may subsequendy directly interact with the given channel in a "membrane delimited" manner, or altematively, they also can activate an intracellular pathway which secondarily modifies the parameters of ion channel activity. The multiplicity of G-proteins and other molecules belonging to corresponding signaling pathways is vast and could account for the pleiotropic action of any transmitter to which they are connected. The detailed molecular mechanisms of these interactions, their compartmentalization in cells as well as dieir regulation and integration in the whole cell biology are still essentially unknown. The main data obtained to further our understanding of this problem are given below. Different G-Protein a-Subunits

Signal transducing heterotrimeric G-proteins are formed of a, P and y subunits. They connect membrane spanning receptors to a variety of effectors such as enzymes or channels. Like small GTPases, they act as switches, cycling between two states, activated and inactivated, depending on the bound nucleotide, GTP or GDP. Interaction of the G-protein with the activated receptor promotes the dissociation of GDP from the a-subunit, the empty site being subsequently occupied by GTP. The binding of GTP leads to the dissociation of the a-GTP complex from the bg heterotrimer and from the receptor. While diversity of the a-subunits may provide some of the required specificity of receptor-G-protein and G-protein effector interactions, it cannot be excuded that the P and y diversity may also contribute to it. Biochemical and Pharmacological Studies of G-Protein Function

Several criteria, either direct or indirect, have to be fulfilled to attribute G-protein intervention to the modulation of ion channels by receptors. The requirement for a G-protein can be explored by injecting into the cell poorly hydrolyzable GTP or GDP analogs. These molecules either activate (GTPyS) or inactivate (GDPpS) the G-proteins of the cells in a relatively irreversible manner. Hence, intracellular injection of GTPyS into the ganglion cells of the Aplysia, have shown, that a GTP-binding protein regulates the opening of potassium channels coupled to transmitter receptors. Addition of GTPyS to the pipette solution in whole-cell patch-clamp, has been shown to mimic some of the neurotransmitter receptor mediated calcium current inhibition in a wide range of excitable tissues. By contrast, GDPpS completely blocked these effects. The use of toxins (cholera toxin (CTx) and PTx) has helped to distinguish between several species of G-proteins. Cholera toxin activates G^ by ADP-ribosylation, whilst other G-proteins, including G^ and G^, have been found to be covalently altered and inactivated by PTx-dependent ADP-ribosylation, resulting in a loss of response to agonist. In this way, the intracellular injection of PTx demonstrated the role of a G-JG^^

442

G.J. LAW, M. RUPNIK, R. ZOREC, P.M. LLEDO, and W.T. MASON

protein in the opening of a receptor-coupled potassium channel, recorded from the ganglion cells of Aplysia. The finding that PTx applied extracellularly or injected direcdy into excitable cells, blocks the inhibitory actions of neurotransmitters demonstrated that in mammalian cells, the coupling of neurotransmitter receptors to ionic channels especially involves G-proteins. Identification of Specific G-proteins Controlling Ionic Channel Activities

Reconstitution experiments using partially purified transmembrane receptors demonstrated that different G-proteins can be associated with these receptors. Thus, bearing in mind: (1) the varied signaling systems affected by transmembrane receptor activation, (2) the diversity of G-protein subunits, and (3) the implications of both a and Py subunits in multiple physiological functions, the question has arisen of which kind of G-protein is involved in the specific coupling of transmembrane receptor to a particular effector. Three different approaches have recently been employed to address these questions. The first one consists in reconstituting the coupling of receptors to ionic channels using purified G^ subunits after inactivation of endogenous G-proteins with PTx. However, reconstitution experiments with purified G-proteins must be cautiously interpreted because the nature and the experimental stoichiometry of the molecules may not reflect the non-experimental situation. Another potential shortcoming of these experiments is the heterogeneity of purified G-protein fractions as these can resist resolution, even after multiple chromatographic steps. Unless the coupled G-protein is perfectly identified, these experiments only give an indication of which G-protein is capable of mediating the channel response and not direct evidence of which one mediates the response in the normal cell. An alternative approach to the identification of the G-proteins involved in transmembrane receptor-induced responses is to use specific antibodies to different G-proteins which disrupt the link between the effector with the receptor. In this respect, the whole-cell recording method affords the advantage of introducing large-molecules such as antibodies into the cell. Hence, although patch pipettes were originally developed for the recording of single channel events, they can be of great advantage for more conventional recordings in the whole-cell mode. For this configuration, the pipette is left attached to the cell after formation of a seal, but the membrane patch under it is ruptured by an additional suction in the pipette. This technique allows the measurement of total membrane currents and/or voltage from the whole-cell. Compared with other cell recording techniques using a dialysis pipette, it is characterized by the very high resistance of the pipette-cell seal. Therefore, the method was initially called "tight-seal whole-cell recording," but in the following description, we use the shorter term "whole-cell recording" abbreviated as WCR. One of the major characteristics of WCR is the access resistance. Because pipette resistance is comparatively low, the clamp circuitry can change the membrane

G-Protein Coupled Receptors and Hormone Secretion

potential quite rapidly. Another consequence of the low-resistance connection between pipette and cell interior is that the cytoplasm is rapidly exchanged with the pipette solution. This can be an advantage in some conditions: (1) for the study of ionic current, the ability to control ionic composition of solutions on both sides of the membrane helps to isolate particular conductances, and (2) for loading cells with different substances. Indeed, the WCR configuration method offers a more gentle way of modifying the intracellular milieu as compared to pressure microinjection. It has been shown that in typical mammalian cells (10-15 jLim diameter) intracellular potassium is exchanged with small mobile cations present in the pipette within 5-10 s. Likewise, larger molecules diffuse into the cells with a time constant in the range of 10 s to 1 min, depending on pipette resistance and on the root of molecular weight. This diffusional exchange offers the possibility of loading cells with substances of interest. We have performed intracellular loading of lactotroph cells with affmity-purified polyclonal antibodies raised against synthetic decapeptides corresponding to the carboxy-terminal sequence of the a-subunits of either G^, 0^3, and G-j 2 (^i^ti-G^^^, anti-Gj3 a^, and anti-Gji 2 ac)- Diffusion of the antibodies into the cells can be demonstrated by fluorescence immuno-histochemistry after patch-clamp recordings (Figure 5). By this means, it can be observed that anti-G^^ but not anti-G^i 2a or anti-Gi3^ antibodies are effective in uncoupling D2 dopamine receptors from both L- and T-type calcium channels in rat pituitary cells, whilst the dopamine-induced increase of both I^ and Ij^ potassium channels was markedly attenuated only by G|3^ antiserum. These findings led to the conclusion that, at least in rat lactotrophs, two different G-proteins are involved in the mechanisms that link D2 receptor activation to voltagegated ionic channels. Other studies, using specific antibodies against G^^, have also highlighted the role of this protein in the coupling of calcium channels to dopaminergic receptors in snail neurons, and to noradrenaline and opiate receptors in neuroblastoma-glioma cell hybrids. However, the diversity of currently recognized GTPases that contain both conserved and divergent sequences will pose a unique challenge to the development of specific tools, such as monoclonal antibodies, to discriminate between the function of each member of the GTPase family. Moreover, there are inherent problems in the use of antibodies, which include the difficulty of introducing them into the cell, the low affinity of many antibodies directed against peptides, and uncertainty as to their specificity for preventing molecular interactions because of their large size. A more accurate method is to selectively impair the synthesis of specific proteins by introducing DNA oUgonucleotides corresponding to the antisense orientation of the respective mRNAs. These antisense probes specifically block the translation of the corresponding message into protein. This strategy has been effective in reducing the concentration of a number of proteins or preventing their induction, provided that the turnover rate of the protein to be blocked is sufficientiy fast with regard to the limited Ufetime of the antisense oligonucleotides themselves.

443

444

G.J. LAW, M . RUPNIK, R. ZOREC, P.M. LLEDO, and W.T. MASON

Figure 5. Fluorescence immunohistochemistry performed after patch-clamp recordings to check whether cells have been properly loaded with antibodies by simple diffusion from the patch pipette into the cells. A rat lactotroph was dialyzed through the patch pipette with an antibody to ao. The cell was recorded for 20 minutes with the electrode filled with anti-ao. Once recorded, the cells were fixed, treated with Triton X-100 to permeabilize the membrane, and then with fluorescein-conjugated porcine immunoglobulins. Cells were viewed under ultraviolet light. Bar = 15 jam.

Microinjection is a more direct and reliable route for the introduction of antisense oligonucleotides into cells and the efficacy of this method has been demonstrated in recent experiments. Hence, it has been shown that microinjection with antisense oligonucleotides successfully blocked the expression of specific G-proteins in the anterior pituitary GH3 cell line. However, the introduction of the oligonucleotides into the cells by nuclear microinjection promoted the loss of a large number of injected cells. To prevent this phenomenon, we have developed a new antisense approach which allows the loading of DNA probes by diffusion from the patch pipette into the cytoplasm. The technique consists of a sequential patch-clamp procedure which allows recordings from the same injected cell, at several day intervals. During the first whole-cell recording, the cells are loaded with the antisense-DNA and then, after various periods of time, the same cells are recorded again to determine the effects of protein absence on a specific cellular response. To check the effects of "knocking out" the expression of a target protein by our sequential patch-clamp procedure, we thus visualized cells by immunofluorescence

G-Protein Coupled Receptors and Hormone Secretion

microscopy after loading with sense or antisense probes. The advantages over antibody quenching of regulatory proteins are two-fold. First, the risks of channel run-down are much lower with the sequential patch clamp technique, since an additional 10 min are required for the dialysis of antibody proteins in the whole-cell recording configuration. Second, oligonucleotides are far easier to produce than antibodies and can be more specific in terms of knocking out a specific function exerted by the target protein. Specificity of the effects of antisense oligonucleotides can be verified by several means. Among them: (1) immunoreactivity can be performed in order to confirm that the reduction of a specific function induced by antisense injection parallels the disappearance of the target protein, (2) rescue experiments can be designed in which the original phenotype is reestablished after microinjection of the purified target protein into antisense-microinjected cells, (3) whether different oligonucleotides complementary to different regions of the mRNA can induce a similar effect has to be tested, (4) control oligonucleotides designed in a sense orientation to the target mRNA should have no effect, (5) pre-hybridization of the antisense to a complementary oligonucleotide which possesses a sequence of the opposite strand which should neutralize the effect of antisense, thus indicating that antisense has to be single-stranded to exert its function, (6) the antisense oligonucleotide has to be able to block the in vitro translation with high specificity, and finally (7) the effect of antisense needs to be found reversible, thus arguing against a toxic effect of the DNA probe. The obvious disadvantages of the sequential patch clamp technique are that it is more time-consuming, technically more demanding, and is endowed with a moderate rate of success at least in normal pituitary cells. The technique should be particularly powerful in cells, for example, neurons, that survive the procedure much better. Effective carriers of oligonucleotides is to independently demonstrate the effectiveness and specificity of the antisense target as an abundantly expressed protein that can easily be quantified by cytochemical means. Obviously, new techniques have still to be applied to facilitate the investigation of the contribution of G-protein to the regulation of ion channels. For example, the use of homologous recombination to "knock-out" a specified gene should be very useful in this respect. Recently, it has been obtained in a cell line devoid of G-2OL which should facilitate the precise dissection of its contribution to the activation of various intracellular signaling pathways. In addition, the use of homologous recombination to obtain transgenic animals, may open the way to a new area of ex-vivo electrophysiology on the tissues of these genetically modified animals.

REFERENCES Aderem, A. (1992). The MARCKS brothers; a family of protein kinase C substrates. Cell 71,713-716. Aridor, M., Rajmilevich, G., Beaven, M.A., & Sagi-Eisenberg, R. (1993). Activation of exocytosis by the heterotrimeric G protein Gi3. Science 262, 1569-1572.

445

446

G.J. LAW, M. RUPNIK, R. ZOREC, P.M. LLEDO, and W.T. MASON

Balch, W.E., Kahn, R.A., & Schwaninger, R. (1992). ADP-ribosylation factor is required for vesicular trafficking between the endoplasmic reticulum and the cis-Golgi compartment. J. Biol. Chem. 267, 13053-13061. Barbacid, M. (1987). Ras genes. Ann. Rev. Biochem. 56, 779-827. Berstein, G., Blank, J.L., Jhon, D.-Y, Exton, J.H., Rhee, S.G., & Ross, E.M. (1992). Phosphohpase C-bl is a GTPase-activating protein for Gq/U, its physiologic regulator. Cell 70, 411-418. Bomsel, M. & Mostov, K. (1992). Role of heterotrimeric G proteins in membrane traffic. Mol. Biol. Cell 3, 1317-1328. Bourne, H.B., Sanders, D.A., & McCormick, F. (1990). The GTPase superfamily: A conserved switch for diverse cell functions. Nature 348,125-132. Bourne, H.R., Sanders, D.A., & McCormick, F. (1991). The GTPase superfamily: A conserved structure and molecular mechanism. Nature 349, 117-126. Bourne, H.R. (1988). Do GTPases direct membrane traffic in secretion? Cell 53, 669-671. Bowman, A.L., Taylor, T C , Melancon, P, & Wilson, K.L. (1992). A role for ADP-ribosylation factor in nuclear vesicle dynamics. Nature 358, 512-514. Brown, H.A., Gutowski, S., Moonaw, C.R., Slaughter, C , & Sternweis, PC. (1993). ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phosphohpase D activity. Cell 75, 1137-1144. Burgoyne, R.D. & Morgan, A. (1993). Regulated exocytosis. Biochem. J. 293, 305-316. Clapham, D.E. (1994). Direct G protein activation of ion channels Ann. Rev. Neurosci. 17,441-464. Cockcroft, S., Thomas, G.M.H., Fensome, A., Geny, B., Cunningham, E., Gout, I., Hiles, I., Totty, N.F., Truang, O., & Hsuan, J.J. (1994). Phosphohpase D: A downstream effector of ARF in granulocytes. Science 263, 523-526. Conklin, B.R., Farfel, Z., Lustig, K.D., Juhus, D., & Bourne, H.R. (1993). Substitution of three amino acids switches receptor specificity of Gqa to that of Gia. Nature 363, 274-276. Conklin, B.R. & Bourne, H.R. (1993). Structural elements of Ga subunits that interact with Gpy receptors and effectors. Cell 73, 631-641. Cotecchia, S., Exum, S., Caron, M.G., & Lefkowitz, R.J. (1990). Regions of the al-adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function. Proc. Natl. Acad. Sci. USA 87, 2896-2900. Dascher, C. & Balch, W.E. (1994). Dominant inhibitory mutants of ARF 1 block endoplasmic reticulum to golgi transport and trigger disassembly of the golgi apparatus. J. Biol. Chem. 269,1437-1448. Davidson, J.S., Eales, A., Roeske, R.W., & Millar, R.P. (1993). Inhibition of pituitary hormone exocytosis by a synthetic peptide related to the effector domain. FEBS Lett. 326, 219-221. Dohlman, H.G., Thomer, J., Caron, M., & Lefkowitz, R.J. (1991). Model systems for the study of seven-transmembrane-segment receptors. Ann. Rev. Biochem 60, 653-688. Donaldson, J.G., Finazzi, D., & Klausner, R.D. (1992). Brefeldin A inhibits golgi membrane-catalyzed exchange of guanine nucleotide onto ARF protein. Nature 360, 350-352. Donaldson, J.G., Cassel, D., Kahn, R.A., & Klausner, R.D. (1992). ADP-ribosylation factor, a small GTP-binding protein is required for binding of the coatomer protein b-COP to Golgi membranes. Proc. Natl. Acad. Sci. USA 89, 6408-6412. Duerson, K., Carroll, R., & Clapham, D, (1993). a-Helical distorting substitutions disrupt coupling between m3 muscarinic receptor and G proteins. FEBS Lett. 324, 103-108. Eckstein, F., Cassel, D., Levkovitz, H., Lowe, M., & Selinger, Z. (1979). Guanosine 5'0-(2Thiodiphosphate) an inhibitor of adenylate cycles stimulation by guaninine nucleotides and fluoride ions. J. Biol. Chem. 254, 9829-9834. Edwardson, J.M., MacLean, C, & Law, G.J. (1993). Synthetic peptides of the rab3 effector domain stimulate a membrane fusion event involved in regulated exocytosis. FEBS Lett. 320, 52-56. Fisher, P.J., Prendergast, E.G., Erhardt, M.R., Urbauer, J.L., Wand, A.J., Sedarous, S.S., McCormick, D.J., & Buckley, P.J. (1994). Calmodulin interacts with amphiphilic peptides composed of all D-amino acids. Nature 368, 651-653.

G'Protein Coupled Receptors and Hormone Secretion

447

Fischer von Mollard, G., Sudhof, T.C., & Jahn, R. (1991). A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature 349, 79-81. Fischer von Mollard, G., Stahl, B., Li, C , Sudhof, T., & Jahn, R. (1994). Rab proteins in regulated exocytosis. Trends in Biol. Sci. 19, 164-168. Gomperts, B.D. (1990). G^: A GTP-binding protein mediating exocytosis. Ann. Rev. Physiol. 52, 591-606. Goud, B., Salminen, A., Walworth, N.C., & Novick, PJ. (1988). A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell 53, 753-768. Goud, B. & McCaffety, M. (1991). Small GTP-binding proteins and their role in transport. Curr. Op. Cell Biol. 3, 626-633. Hamm, H.E., Deretic, D., Hofman, K.P, Schleicher, A., & Kohl, B. (1988). Site of G protein binding to rhodopsin mapped with synthetic peptides from the a subunit. Science 241, 832-835. Helms, J.B. & Rothman, J.E. (1992). Inhibition by brefeldin A of a golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF. Nature 360, 352-354. Hess, S.D., Doroshenko, PA., & Augustine, G.J. (1993). A functional role for GTP-binding proteins in synaptic vesicle cycling. Science 259, 1169-1172. Higashijima, T., Uzu, S., Nakajima, T., & Ross, E.M. (1988). Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins. J. Biol. Chem. 263, 6491-6494. Higashijima, T., Bumier, J., & Ross, E.M. (1990). Regulation of Gi and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines. J. Biol. Chem. 265, 14176-14186. Hirai, Y, Yasuhara, T., Yoshida, H., Nakajima, T., Fujino, X, & Kitada, M. (1979). Chem. Pharm. Bull. (Tokyo) 27, 1942-1944. Ikezu, T., Okamoto, T., Ogata, E., & Nishimoto, I. (1992). Amino acids 356-372 constitute a Gj-activator sequence of the a2-adrenergic receptor and have a Phe substitute in the G protein-activator sequence motif. FEBS Lett. 311, 29-32. Iniguez-Lluhi, J., Kleuss, C , & Oilman, A.G. (1993). The importance of G-protein py subunits. Trends in Cell Biol. 3, 230-235. Johannes, L., Lledo, P-M., Roa, M., Vincent, J.-D., Henry, J.-P, & Darchen, F (1994). The GTPase Rab3a negatively controls calcium dependent exocytosis in neuroendocrine cells. EMBO J. 13, 2029-2037. Kahn, R.A., Randazzo, P, Sefafmi, T., Weiss, O., Rulka, C , Clark, J., Amherdt, M., Roller, P, Orci, L., & Rothman, J.E. (1992). The amino terminus of ADP-ribosylation factor (ARF) is a critical determinant of ARF activities and is a potent and specific inhibitor of protein transport. J. Biol. Chem. 267,13039-13046. Kahn, R.A., Yucel, J.K., & Malhotra, V. (1993). ARF signaling: A potential role for phospholipase D in membrane traffic. Cell 75, 1045-1048. Kleuss, C , Scherubl, H., Hescheler, J., Schultz, G., & Wittig, B. (1993). Selectivity in signal transduction determined by y units of heterotrimeric G proteins. Science 259, 832-834. Kleuss, C , Scherubl, H., Hescheler, J., Schultz, G., & Wittig, B. (1993). Different p-subunits determine G-protein interaction with transmembrane receptors. Nature 358, 424-426. Koch, G., Haberman, B., Mohr, C , Just, L, & Akories, K. (1991). Interaction of mastoparan with the low molecular mass GTP-binding proteins rho/rac. FEBS Lett. 291, 336-340. Law, G.J., Northrop, A.J., & Mason, WT. (1993). rab3-Peptide stimulates exocytosis from mast cells via a pertussis toxin-sensitive mechanism. FEBS Lett. 333, 56-60. Lenhard, J.M., Kahn, R.A., & Stahl, P (1992). Evidence for ADP-ribosylation factor (ARF) as a regulator of m vitro endosome-endosome fusion. J. Biol. Chem. 267, 13047-13052. Leyte, A., Barr, FA., Kehlenbach, R.H., & Huttner, WB. (1992). Multiple trimeric G proteins on the trans-Golgi network exert stimulatory and inhibitory effects on secretory vesicle formation. EMBO J. 11,4795-4804.

448

G.J. LAW, M. RUPNIK, R. ZOREC, P.M. LLEDO, and W.T. MASON

Li, G., Regazzi, R., Balch, W.E., & WoUheim, C.B. (1993). Stimulation of insulin release from permeabilized HIT-T15 cells by a synthetic peptide corresponding to the effector domain of the small GTP-binding protein rab3. FEES Lett. 327, 145-149. Lillie, T.H.W. & Gomperts, B.D. (1992). Nucleotides and divalent cations as effectors and modulators of exocytosis in permeabilized rat mast cells. Phil. Trans. R. Soc. Lond. B. 336, 25-34. Lindau, M. & Neher, E. (1988). Patch-clamp techniques for time-resolved capacitance measurements in single cells. Pfluegers Archiv. 411,137-146. Lledo, RM., Vernier, R, Vincent, J., Mason, W.T., & Zorec, R. (1993). Inhibition of Rab3b expression attenuates Ca "*" dependent exocytosis in rat anterior pituitary cells. Nature 364, 540-544. McKieman, C.J., Brondyk, W.H., & Macara, I.G. (1993). The rab3 GTPase interacts with multiple factors through the same effector domain. Mutational analysis of cross-linking of rab3a to a putative target protein. J. Biol. Chem. 268, 24449-24452. Milbum, M.V., Tong, L., DeVos, A.M., Brunger, A., Yamaizumi, Z., Nishimura, S., & Kim, S-H. (1990). Molecular switch for signal transduction: Structural differences between active and inactive forms of protooncogenic ras proteins. Science 247, 939-945. Miller, S.G., Camell, L., & Moore, H-RH. (1992). Post-golgi membrane traffic: Brefeldin A inhibits export distal golgi compartments to the cell surface but not recycling. J. Cell Biol. 118,267-283. Montecucco, C. & Schiavo, G. (1993). Tetanus and botulism neurotoxins: A new group of zinc proteases. Trends in Biochem. Sci. 18, 324-329. Morgan, A. & Burgoyne, R.D. (1993). A synthetic peptide of the N-terminus of ADP-rybosilation factor (ARF) inhibits regulated exocytosis in adrenal chromaffin cells. FEBS Lett. 329, 121-124. Neher, E. & Marty, A. (1982). Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells. Proc. Natl. Acad. Sci. USA 79, 6712-6716. Munch G., Dees, C., Hekman, M., & Palm, D. (1991). Multisite contacts involved in coupling of the p-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein. Eur. J. Biochem 198, 357-364. Oberhauser, A.F., Monk, J.R. Balch, W.E., & Fernandez, J.M. (1992). Exocytotic fusion is activated by Rab3a peptides. Nature 360, 270-273. Okamoto, T, Katada, T, Murayama, Y, Ui, M., Ogata, E., & Nishimoto, I. (1990). A simple structure encodes G protein-activating function of the IGF-II/mannose 6-phosphate receptor. Cell 62, 709-717. Okano, K., Monck, J.R., & Fernandez, J.M. (1993). GTP-y-S stimulates exocytosis in patch-clamped rat melanotrophs. Neuron 11, 165-172. Oppi, C , Wagner, T, Crisari, A., Camerini, B., & Valentini, G.RT. (1992). Attenuation of GTPase activity of recombinant Goa by peptides representing sequence permutations of mastoparan. Proc. Natl. Acad. Sci. USA. 89, 8268-8272. Noel, J.R, Hamm, H.E., & Sigler, RB. (1993). The 2.2A crystal structure of transducin-a complexed with GTPyS. Nature 366, 654-663. Padfield, P.J., Balch, W.E., & Jamieson, J.D. (1992). A synthetic peptide of the rab3A effector domain stimulates amylase release from permeabilized pancreatic acini. Proc. Natl. Acad. Sci. USA 89, 1656-1660. Pfeffer, S.R. (1992). GTP-binding proteins in intracellular transport. Trends Cell Biol. 2,41-46. Plutner, H., Schwaninger, R., Pind, S., & Balch, W.E. (1990). Synthetic peptides of the rab effector domain inhibit vesicular transport through the secretory pathway. EMBO J. 9, 2375-2383. Polakis, P. & McCormack, F. (1993). Structural requirements for the interaction of p21 ras with GAP, exchange factors, and its biological effector target. J. Biol. Chem. 268, 9157-9160. Regazzi, R., Ullrich, S., Kahn, R.A., & Wollheim, C.B. (1991). Redistribution of ADP-ribosylation factor during stimulation of permeabilized cells with GTP analogs. Biochem J. 275, 639-644. Rupnik, M. & Zorec, R. (1994). Heterotrimeric GTP-binding proteins control Ca^"*"-independent secretory activity of rat melanotrophs. J. Physiol. 475, 142P.

G'Protein Coupled Receptors and Hormone Secretion

449

Salminen, A. & Novick, P.J. (1987). A ras-like protein is required for a post-Golgi event in yeast secretion. Cell 49, 527-538. Savarese, T.M. & Fraser, CM. (1992). In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors. Biochem. J. 283, 1-19. Schaber, M.D., Garsky, V.M., Boylan, D., Hill, W.S., Scolnick, E.M., Marshall, M.S., Sigal, I.S., & Gibbs, J.B. (1989). Ras interaction with the GTPase-activating protein (GAP). Proteins: structure, function & genetics 6, 306-315. Senyshyn, J., Balch, W.E., & Holz, R.W. (1992). Synthetic peptides of the effector-binding domain of rab enhance secretion from digitonin-permeabilized chromaffin cells. FEBS Lett. 309, 41-46. Shirataki, et al. (1993). Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin. Mol. Cell. Biol. 13, 2061-2068. Sigal, I.S., Gibbs, J.B., D'Alonzo, J.S., & Scholnick, E.M. (1986). Identification of effector residues and a neutralising epitope of Ha-ras-encoded p21. Proc. Natl. Acad. Sci. USA 83,4725-4729. Sikdar, S.K., Zorec, R., Brown, D., & Mason, W.T. (1989). Dual effects of G-protein activation on Ca-dependent exocytosis in bovine lactotrophs. FEBS Lett. 253, 88-92. Simmons, K. & Zerial, M. (1993). Rab proteins and the road maps for intracellular transport. Neuron 11,789-799. Srivastava, S.K., Varma, T.K., Sinha, A.C., & Singh, U.S. (1994). Guanosine 5'-(Y-thio) triphosphate (GTPyS) inhibits phosphorylation of insulin receptor and a novel GTP-binding protein, Gir, from human placenta. FEBS Lett. 340, 124-128. Stouten, P.F.W., Sander, C , Wittinghofer, A., & Valencia, A. (1993). How does the switchl 1 region of G-domains work? FEBS Lett. 320, 1-6. Stow, J.L., Bruno de Almeida, J., Narula, N., Holtzman, E.J., Ercolani, L., & Ausiello, D.A. (1991). A heterotrimeric G protein, G^^i^^ on golgi membranes regulates the secretion of a heparan sulphate proteoglycan in LLC-PKl epithelial cells. J. Cell. Biol. 114, 1113-1124. Strader, CD., Sigal, LS., & Dixon, R.A.F. (1989). Structural basis of P-adrenergic receptor function. FASEB J. 3, 1825-1832. Stutchfield, J. & Cockcroft, S. (1993). Correlation between secretion and phospholipase D activation in differentiated HL60 cells. Biochem. J. 293, 649-655. Sullivan, K.A., Miller, R.T., Masters, S.B., Biederman, B., Heiderman, W., & Bourne, H.R. (1987). Identification of receptor contact site involved in receptor-G protein coupling. Nature 330, 758-760. Takai, Y, Kaibuchi, K., Kikuchi, A., & Kawata, M. (1992). Small GTP-binding proteins. Int. Rev. Cytol. 133, 187-229. Tatham, P.E.R. & Gomperts, B.D. (1991). Late events in regulated exocytosis. Bioessays 13, 397-401. Tomita, U., Takanashi, K., Ikenaka, K., Kondo, T, Fujimoto, I., Aimoto, S., Mikoshiba, K., Ui, M., & Katada, T. (1991). Direct activation of GTP-binding protein by venom peptides that contain cationic clusters within their alpha-helical structures. Biochem. Biophy. Res. Comm. 178, 400406. Van Dop, C , Yamanaka, G., Steinberg, F, Sekura, R., Manclark, C.R., Stryer, L., & Bourne, H.R. (1984). ADP-ribosylation of transducin by pertussis toxin blocks the light-stimulated hydrolysis of GTP and cGMP in retinal photoreceptors. J. Biol. Chem. 259, 23-25. Vitale, N., Mukai, H., Rouot, B., Thierse, D., Aunis, D., & Bader, M.F. (1993). Exocytosis in chromaffin cells: Possible involvement of the heterotrimeric GTP-binding protein Go. 268, 14715-14723. Voss, T, Wallner, E., Czemilofsky, A.P, & Freissmuth, M. (1993). Amphipathic a-helical structure does not predict the ability of receptor-derive synthetic peptides to interact with guanine nucleotidebinding regulatory proteins. J. Biol. Chem. 268, 4637-4642. Wakamatsu, K., Okada, A., Miyazawa, T., Ohya, M., & Higashijima, T. (1992). Membrane-bound conformation of mastoparan-X, a G-protein-activating peptide. Biochemistry 31, 5654-5660. Weingarten, R., Ransnas, L., Mueller, H., Sklar, L.A., & Bokoch, G.M. (1990). Mastoparan interacts with the carboxyl terminus of the a subunit of Gi. J. Biol. Chem 265, 11044-11049.

450

G.J. LAW, M. RUPNIK, R. ZOREQ P.M. LLEDO, and W.T. MASON

Zhang, C.J., Rosenwald, A.G., Willingham, M.C., Skuntz, S., Clark, J., & Kahn, R.A. (1994). Expression of a dominant allele of human ARFl inhibits membrane traffic in vivo. J. Cell Biol. 124,289-300. Zeuzem, S., Zimmermann, P., & Schulz, I, (1991). Guanosine nucleotides modulate the inhibitory effect of brefeldin A on protein secretion. FEES Lett. 288,143-146. Zorec, R., Henigman, R, Mason, W.T., & Kordas, M. (1991). Electrophysiological study of hormone secretion by single adenohypophyseal cells. Methods in Neurosci. 4, 194-209.