Siva R. P. Gudi Ph.D. and John A. Frangos Ph.D. Department of Bioengineering, University of California-San Diego, La Jolla, CA 92093. Aim: Guanyl nucleotide binding proteins (G-proteins) are a group of proteins which are central elements in signal transduction pathways and show great complexity in structure and function. G-proteins have heterotrimeric structure consisting of separate a, b and g subunits. The a subunit has intrinsic GTPase activity and in inactive state with bound GDP, it complexes with bg subunits. Upon stimulation with GTP replacing GDP, a subunit dissociates from bg complex causing a downstream effector activation in the signal transduction pathway (1). The main objective of the present study is to characterize G proteins involved in signal transduction mechanisms induced by various physical and chemical agonists in mammalian cells (endothelial cells, fibroblasts). Photoaffinity labeling is one of the effective techniques used to identify G proteins in plasma membranes (2). We have adopted this methodology to studies in whole cells by preloading cells with [32P] labeled photoactive GTP analog, GTPg-4-Azidoanilide (GTPgAA). We used digitonin to load the cells with photoprobe for shorter periods (3 min) before they were subjected to stimulation. Photoactivatable azido group substitution allows nucleotide to be covalently cross-linked by UV exposure once bound to the active site. The 32P label on GTP photoprobes facilitates the identification of GTP binding proteins (3). With this method it should be possible to selectively stimulate the release of bound GDP, followed by the binding of GTPgAA or GTPg-Acetanilide (GTPgAcA), only in the species of G protein(s) which interact with the agonist-occupied under study.
Experimental Protocol and Results: Exp. 1: G-protein photolabeling in plasma membranes: Plasma membrane preparations were photolabeled with photoactivatable analogs [g-32P]GTPgAA (4)or GTPgAcA (5). Membranes were diluted in 10 mM Tris, 0.1 mM EDTA (pH 7.5) to a concentration of 2.5 mg/ml. A 20 µl aliquot of assay buffer (3x) {HEPES, 30 mM (pH 7.4); NaCl, 10 mM; EDTA, 0.1 mM; MgCl2, 5 mM; GDP, 3 µM and Benzamidine, 1 mM (final conc. in assay)} was added to 20 µl of diluted plasma membranes with or without 6 µl of receptor agonist(s) in a microfuge tube. Tubes were then pre-incubated for 3 min at 30oC and cooled to 4oC by transferring over ice. [g32P]GTPgAA or [g-32P] GTPgAcA (1 µCi/ tube) was added in 10 ml at a final conc. of 10 to 50 nM in final assay volume of 60 µl. Tubes were incubated for a further 3 min at 30oC and the assay was terminated by cooling the samples to 4oC by moving over ice. Unbound photoprobe was removed by centrifugation of the tubes at 100,000 x g for 10 min at 4oC. Supernatant was carefully discarded and the pellets were resuspended in a modified version of above assay buffer (with the addition of 2 mM DTT and without GDP). DTT presumably prevents nonspecific cross-linking of GTP analog during UV exposure. Membrane suspensions were then irradiated at 4oC for 1 min at a distance of 3 cm with a hand held UV lamp (254 nm). Membrane samples were centrifuged and the pellets were dissolved in 20 µl of SDS-PAGE sample buffer to separate by electrophoresis and analyzed by exposure to X-ray film for 3 days (Fig 1) and 5 days (Fig 2).
Fig. 1: Photolabeling of G-proteins with [g32P]GTPgAcA in endothelial cell plasma membranes: Autoradiogram of experiment 1. A, B, C and D are membrane samples stimulated with various agonists. Fig. 2: G-Proteins photolabeled with [g32P]GTPgAA in endothelial cell plasma membranes: Autoradiogram of the experiment 1. A and C unstimulated and B stimulated samples
Exp. 2: G-Protein photolabeling in whole cells: Cells (HUVECs / Fibroblasts) were washed with Hanks Balanced Salt Solution to remove the growth medium (with serum) and incubated in medium without serum for additional 2 to 3 hr in a CO2 incubator before starting the experiment. Cells were preincubated with 20 µM Digitonin and the [32P] GTP analog (10µCi/106 cells in monolayer) in the medium for 3 min. The total volume of incubation mixture was 100 µl (enough to overlay the cells on the glass slide) Cells were rinsed off with the incubation medium and subjected to stimulation {Stretch, Fluid Flow, Bradykinin or Angiotensin II}. Exposure to UV was used as a stimulus termination. Cells were exposed to UV light (short wave, 254 nm) at a distance of 3 cm on ice for 1 min. Before exposure to UV, cells were rinsed with phosphate buffered saline containing DTT (2 mM) to inactivate unbound photoactivatable GTP analog. Cells were harvested using SDS-PAGE sample buffer and the samples were separated by electrophoresis and analyzed by exposure to X-ray film (data not shown).
Exp. 3: Identification of photolabeled G-Proteins by immunoprecipitation: In another experiment the photolabeled G-Proteins were further identified by immunoprecipitation. Experiment 2 was repeated as described and after UV cross-linking, the cells were lysed in ice-cold RIPA buffer {50 mM Tris-HCl (pH 7.4), NaCl (150 mM), Nonidet P-40 (1%), Sodium deoxycholate (0.25%), EGTA (1 mM), PMSF (1 mM), Na3VO4 (1mM ), NaF(1 mM), Leupeptin, Aprotonin, Pepstatin (each), 1 µg/ml}. A B C D E F G H I
Fig. 3: G-protein photolabeling by [g32P]GTPgAA in rat cardiac fibroblasts: A and B: Control, C, D, E, F: stimulated, G and H: Angiotensin II (10-9M) and I: Bradykinin (10-6M).
Cell lysates were centrifuged at 100, 000 xg to clear the lysates. Polyclonal antibodies to Ga subunits were added and incubated overnight at 4°C in a rotary shaker. Protein A coupled trisacryl beads were added to collect the immunocomplexes. The immunoprecipitates were washed with ice-cold RIPA buffer (5x). Electrophoresis sample buffer was added to the precipitates. Samples were separated by SDS-PAGE and exposed to X-ray film for 6 days. Protein was determined by the Bradford procedure (6) with BSA as standard. DISCUSSION [32 P]GTPgAA is stored in absolute methanol at -20oC until ready to use. Aliquot of the probe was dried in a speedvac and the dried probe was dissolved in the incubation medium. [32 P]GTPg-AcA is relatively stable in aqueous buffers. Whenever pure protein preparations or membrane preparations were used reducing agents like DTT were removed before the experiment. Radiolabeled photoaffinity analogs GTPgAcA (Fig.1) and GTPgAA (Fig.2) are readily incorporated into proteins with molecular mass of 42 kDa only when plasma membranes purified from human endothelial cells were stimulated with agonists. Similar, consistent labeling was also observed with whole cells. When permeabilized rat fibroblasts were stimulated with a variety of agonists (Fig. 3), GTP analog gave similar labeling patterns of 40-45 kDa proteins which corresponds to a subunits of heterotrimeric G-proteins (Fig. 3). In order to establish that photoprobes are incorporated into Ga subunits, we immunoprecipitated the endothelial cell lysates with antibodies specific to Ga subunits after agonist specific photolabeling. Immunoprecipitation followed by SDS-PAGE analysis resulted in labeling of Gq as well as Gi3, each with a molecular mass of 42 kDa. A common problem of using non-hydrolyzable analog of GTP to activate G-proteins is that the binding of these analogs is too rapid to allow agonist induced increases in the binding rate to be measured. As with the activating nucleotides GTPgS and GPPNP, GTPgAA is resistant to hydrolysis and produces persistent activation of G-proteins. However, in the absence of agonist the rate of binding of GTPgAA to the G-protein a subunit seems to be considerably slower than that of both GTPgS and GPPNP. Photoaffinity cross-linking is an effective technique which together with other functional assays can be used to determine the activation of proteins in cells. This report provides a detailed protocol for labeling G proteins in plasma membrane preparations as well as in whole cells with photoaffinity GTP analogs. It can be expected that these photoaffinity labels will serve as useful tools in studies of cellular phenomena mediated by guanine nucleotide-binding proteins, such as signal transduction, secretion, endocytosis and cellular growth control. The methodology described here can be easily extended to other nucleotide binding proteins.
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Additional Publications from the Laboratory of Dr. John A Frangos, Ph.D.
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