Stephen J. Taylor

Nonradioactive determination of Ras-GTP levels using activated ras interaction assay

Publisher Summary This chapter describes the nonradioactive determination of Ras- guanosine-5-triphosphate (GTP) levels using activated ras interaction assay. In their activated, GTP-bound forms the Ras proteins interact with a variety of intracellular effector proteins to initiate signaling pathways that contribute to cell proliferation, differentiation, or death, depending on cellular context. The level of Ras activation (the fraction of total Ras molecules in the GTP-bound form) and the duration of Ras activation (the length of time a given fraction is in the GTP-bound form) are controlled by the opposing actions of exchange factors that stimulate guanosine-diphosphate (GDP) for GTP exchange and GTPase-activating proteins (GAPs), which stimulate the intrinsic GTPase activity of Ras. The first generation of Ras—GTP assays directly measured the ratio of GTP to GDP bound to Ras extracted from cells. If trying to detect a low level of Ras activation it may be necessary to optimize assay conditions using normally activated—that is, nonmutated, Ras.

Phosphorylation of the Src substrate Sam68 by Cdc2 during mitosis

Sam68 (Src-associated in mitosis) is an SH3 (Src-homology 3), SH2 (Src-homology 2), and RNA binding protein which associates with and is tyrosine phosphorylated by wild-type and activated forms of c-Src in a mitosis-specific manner. We now show that Sam68 immunoprecipitated from either HeLa S3 or NIH3T3 cells is phosphorylated on threonine residues exclusively during mitosis as well as on serine residues during both interphase and mitosis. Recombinant Sam68, expressed as a glutathione S-transferase (GST) fusion protein, was phosphorylated on threonine and serine residues after incubation with mitotic lysates several-fold more extensively than after incubation with unsynchronized lysates. Cdc2 was identified as the kinase responsible for the mitotic threonine phosphorylation by (1) immunodepletion of the mitotic Sam68 kinase from cell lysates with anti-Cdc2 antibodies, (2) inhibition of Sam68 phosphorylation in vitro and in vivo by the cyclin-dependent kinase inhibitor olomoucine and (3) phosphorylation of Sam68 by purified Cdc2. These data demonstrate that Sam68 is a direct target of Cdc2 and may therefore mediate some of its biological effects during mitosis.

Cell signalling through phospholipid breakdown

There is much evidence that G-proteins transduce the signal from receptors for Ca2+-mobilizing agonists to the phospholipase C that catalyzes the hydrolysis of phosphoinositides. However, the specific G-proteins involved have not been identified. We have recently purified a 42 kDa protein from liver that activates phosphoinositide phospholipase C and cross-reacts with antisera to a peptide common to G-protein α-subunits. It is proposed that this protein is the α-subunit of the G-protein that regulates the phospholipase in this tissue.

ALTERATIONS IN VASOPRESSIN AND ANGIOTENSIN II RECEPTORS AND RESPONSES DURING CULTURE OF RAT LIVER CELLS

Vasopressin and angiotensin II binding and responses were studied in hepatocytes in primary culture for 4 h and 24 h. After 24 h of culture, angiotensin II was completely ineffective in elevating cytosolic [Ca2+], whereas the maximum [Ca2+] response to vasopressin was decreased by 66% and the sensitivity to the hormone was decreased approx. 20-fold compared with values after 4 h of culture. The dissociation constant (KD) for vasopressin binding to the cells was not significantly changed during 24 h of culture, but the Bmax was decreased by 63% compared with 4 h of culture. There was also no change in the KD for angiotensin II binding from 4 h to 24 h, but the Bmax was decreased by 90%. After 24 h of culture, there was no change in the plasma membrane concentration of phosphatidylinositol 4,5-bisphosphate or in the basal cell concentration of inositol trisphosphate. However, the trisphosphate did not increase with 100 nM angiotensin II and the response to 100 nM vasopressin was reduced by 66% compared with that at 4 h. The effect of guanosine 5-(3-O-thiol) triphosphate on the polyphosphoinositide phospholipase C activity of liver cell plasma membranes was also measured. There was no decrease in the degree of stimulation of the phospholipase by this nucleotide after 24 h of culture. It is concluded that the loss of vasopressin and angiotensin II responses in cultured liver cells is due in part to changes in receptors and also in their coupling to a guanine nucleotide binding protein.

Formation of the high-affinity agonist state of the α1-adrenergic receptor at cold temperatures does not require a G-protein

Two methods were employed to uncouple hepatic α1‐adrenergic receptors from their associated G‐protein (termed Gp) in order to determine wether locking of the α1‐receptor in a high‐affinity agonist state at cold temperatures (2°C) represents formation of a ternary complex. Uncoupling is defined as the inability to observe the GppNHp‐sensitive, high‐affinity agonist state of the receptor in [3H]prazosin competition binding studies performed at 25°C. The first method for achieving uncoupling involved brief alkalinization and resulted in greater 95% loss of several G‐proteins. The second method involved proteolytic cleavage of either part or all of the α1‐receptor coupling domain from the binding domain. Following either treatment, receptors were converted to the high‐affinity agonist state at 2°C. Thus, while formation of the high‐affinity state of the receptor at higher temperatures may require Gp, formation of this state at 2°C does not require Gp or even the entire α1‐adregenic receptor.

Purification of the hepatic vasopressin receptor using a novel affinity column

A vasopressin receptor was purified, using a novel affinity column, from rat liver plasma membranes treated with guanosine 5-(3-O-thio)triphosphate and solubilized with 0.8% cholate. Incubation of the membranes with the GTP analogue resulted in a dissociation of the receptor-guanine nucleotide regulatory protein complex. This manipulation, although resulting in a low-affinity state of the receptor, facilitated purification. The solubilized receptor was assayed using a new reconstitution procedure in which the soluble extracts were inserted into lipid vesicles composed of phosphatidylcholine and phosphatidylinositol. The receptor was purified by sequential chromatography on Q-Sepharose and hydroxyapatite. The use of a novel affinity column, a V1-vasopressin antagonist-agarose, resulted in a near-homogeneous preparation of a protein which exhibited an Mr = 58,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of purified receptor, as well as crude membrane preparations cross-linked to [125I]arginine vasopressin, also revealed a protein band with an approximate Mr = 58,000. These findings indicate that V1-antagonist affinity chromatography should be useful for purifying adequate amounts of the receptor for studies of structure and function.

The G-Proteins Regulating Phosphoinositide Breakdown

The stimulation of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis is a widespread cellular response to many hormones, growth factors and neurotransmitters (Berridge 1987). It is catalyzed by a phospholipase C (PLC) and yields two signaling molecules: inositol 1,4,5-trisphosphate (IP3) which releases Ca2+ from stores in the endoplasmic reticulum, and 1,2-diacylglycerol (DAG) which activates protein kinase C. The growth factors activate the PLC through the tyrosine kinase activity of their receptors (Kriz et al 1990), whereas the other agonists act through guanine nucleotide-binding regulating proteins (G-proteins). Despite the recognition several years ago that G-proteins were involved in the regulation of PLC, some of these G-proteins have only recently been identified (Taylor et al 1990, Smrcka et al 1991, Blank et al 1991). These G-proteins are insensitive to pertussis toxin, but it is clear that toxin-insensitive G-proteins are also involved in the regulation of PLC in some tissues (Exton 1988).

HEPATIC VASOPRESSIN RECEPTOR: A KEY RECEPTOR OF PHOSPHOINOSITIDE METABOLISM

It is generally accepted that many drugs, hormones and neurotransmitters elicit responses in cells by interacting with specific receptors located in the plasma membrane. A number of receptors for hormones and neurotransmitters have been purified to homogeneity and functionally reconstituted. Most of them mediate agonist actions by interacting with effector systems to modulate the intracellular levels of second messengers via members of a family of guanine nucleotide binding proteins (G proteins).