Subcellular Localization and Quantitation of the Major Neutrophil Pertussis Toxin Substrate

The subcellular distribution of G protein subunits in the neutrophil was examined. Cells were nitrogen cavitated and subcellular organelles fractionated on discontinuous sucrose gradients. The presence of GTP-binding regulatory protein (G protein) alpha and beta/gamma subunits in each organelle was determined using three methods of analysis: specific binding of guanine nucleotide, ADP ribosylation by pertussis toxin, and immunoblot analysis with subunitspecific G protein antibodies. Both plasma membrane and cytosolic G protein components were detected. In contrast, neither the specific nor the azurophilic granules contained detectable G protein. Based on the ability of exogenous G protein beta/gamma subunits to increase the ADP ribosylation of the cytosolic form of G protein and upon the hydrodynamic behavior of the cytosolic protein, it is likely that this represents an uncomplexed G protein alpha subunit. Proteolytic mapping with Staphylococcus aureus V8 protease suggests the soluble alpha subunit is from G,, the major pertussis toxin substrate of human neutrophils. Using quantitative analysis, the levels of the 40-kD G protein alpha subunit and of the 35/36-kD beta subunit in the neutrophil membrane were determined. T r~E GTP-binding regulatory proteins (G proteins) j consist of a family of highly homologous proteins which serve to couple various membrane-bound hormone, neurotransmitter, and chemotactic factor receptors to their cellular effector systems. At least five such proteins have been purified and characterized, including the following: Gs and G~, the stimulatory and inhibitory regulators of adenylate cyclase (5, 38); transducin, which couples rhodopsin to the cGMP phosphodiesterase in rod outer segments (18); Go, a G protein abundant in brain whose function is as yet unclear but which has been demonstrated to interact with muscarinic cholinergic receptors (9, 37); and G,, a G protein found in neutrophils which may couple chemotactic factor receptors to a phospholipase C (12, 29). In addition, several other potential G proteins have been described (7, 16, 41). The G proteins are structurally similar, being heterotrimers consisting of distinct alpha and very similar, if not identical, beta/gamma subunits. The alpha subunits in each G protein contain the sites for binding guanine nucleotides and fluoride (5, 26), as well as sites for ADP ribosylation by various bacterial toxins (4, 27). Alpha subunits of at least two G proteins (G~ and Go) have been shown to behave as soluble proteins in vitro, maintaining behavior as monomeric species in the absence of detergents (36). The beta/gamma subunits form a relatively hydrophobic complex which may I. Abbreviations used in this paper: DIFP, diisopropylfluorophosphate; G proteins, GTP-binding regulatory proteins; GTPTS, guanosine 5'-gamma (3-0-thio) triphosphate. be involved in coupling the alpha subunits to membrane receptors (9) and, potentially, the membrane itself. The ability of beta/gamma to promote the association of the alpha subunit with artificial phospholipid vesicles has been demonstrated (36). While the G proteins largely appear to exist as membrane proteins, requiring detergents to extract them from membranes, there are indications that exceptions to this generalization exist. In particular, transducin has been shown to behave as a peripherally bound membrane protein at physiologic ionic strength (19). The transducin alpha subunit can be released from the membrane and become soluble by treatment with light (i.e., the receptor "agonist") and GTP. Under conditions of low ionic strength, both light and GTP are individually effective in releasing the holoprotein complex from the membrane. The regulation of a soluble cGMP phosphodiesterase by transducin may require formation of the soluble, activated species of Tn-alpha (39). Several other reports suggest the possibility that other G proteins may be able to exist in a soluble form, including Gs (21, 32) and some of the pertussis toxin substrates (12, 23). In this report, we analyze in detail the subcellular distribution of G protein subunits in the human neutrophil. A cytoplasmic pertussis toxin substrate is described and characterized. Proteolytic mapping identifies the cytosolic protein as identical to the 40-kD alpha subunit of G, that exists in neutrophil membrane and clearly distinct from the 41-kD alpha subunit of G~. The cytoplasmic form of G. appears to represent the alpha subunit of the protein uncomplexed from the beta/gamma subunit. Quantitative estimates of the levels © The Rockefeller University Press, 0021-9525/88/06/1927/10 $2.00 The Journal of Cell Biology, Volume 106, June 1988 1927-1936 1927 on N ovem er 6, 2017 jcb.rress. org D ow nladed fom