John R. Didsbury

Chemoattractant stimulus-response coupling

Phagocytes migrate from the circulation to accumulate at sites of inflammation in response to proinflammatory mediators. A variety of chemoattractants have been identified which stimulate the accumulation and activation of phagocytic leukocytes (Table I). The migratory responses of leukocytes occur at chemoattractant concentrations at least 20-fold lower than the concentration required to stimulate a secretory response. At high concentrations, chemoattractants initiate potentially cytotoxic or microbicidal secretory responses by leukocytes through the degranulation of storage vesicles and the production of toxic oxygen products. The differential nature of these responses is correlated with specific pharmacologic manipulations and biochemical mechanisms. Chemoattractants induce rapid and sequential alterations of intracellular ion concentrations and phospholipid metabolism.

Both stimulatory and inhibitory GDPGTP exchange proteins, smg GDS and rho GDI, are active on multiple small GTP-binding proteins☆

Six peaks of small GTP-binding proteins (G proteins) were separated by column chromatographies from the cytosol fraction of the differentiated HL-60 cells: two peaks of rho p21, one peak of smg/rap1 p21, two peaks of rac1 p21, and one peak of an unidentified small G protein with a Mr of about 20,000 (20 KG). smg GDS, previously thought to be a stimulatory GDP/GTP exchange protein for smg p21, Ki-ras p21, and rho p21, but not for Ha-ras p21 or smg p25A, was also active on rac1 p21. rho GDI, previously thought to be an inhibitory GDP/GTP exchange protein specific for rho p21, was also active on rac1 p21. These results indicate that both smg GDS and rho GDI are active on multiple small G proteins.

Isoprenylation of the low molecular mass GTP-binding proteins rac 1 and rac 2 : possible role in membrane localization

Ras proteins can be modified at their COOH-terminal cysteine in the motif Cys-Ali-Ali-Xaa by a farnesyl isoprenoid. This modification is essential for membrane association and biological activity of ras proteins. A similar COOH-terminal amino acid sequence, Cys-Xaa-Ali-Xaa, exists in the ras-related GTP-binding proteins rac 1 and rac 2. To determine whether these proteins were similarly modified, COS cells were transfected with rac 1 and rac 2 cDNA and expressed proteins were labeled with [3H]mevalonic acid. We report here that both rac 1 and rac 2 are post-translationally modified by addition of an isoprenoid group, the likely site of which is the COOH-terminal cysteine. Isoprenylation was found only in racs associated with particulate cell fractions, suggesting that this modification may be associated with membrane localization of the proteins. These data specifically identify mammalian low molecular mass GTP-binding proteins other than ras that undergo post-translational modification and further define the COOH-terminal consensus sequence, Cys-Ali-Ali-Xaa, as an isoprenylation signal. This sequence may identify a larger family of low molecular mass GTP-binding proteins which are isoprenylated.

Differential regulation of cAMP by endogenous versus transfected fornylpeptide chemoattractant receptors: Implications for Gi-coupled receptor signaling

Endogenous neutrophil formylpeptide receptors do not inhibit adenylylcyclase activation. The ability of a cloned and transfected human formylpeptide receptor to mediate the inhibition of adenylylcyclase was assessed in the human embryonic kidney 293 TSA cell line. Inclusion of 1 microM fMetLeuPhe resulted in a ca. 50% inhibition of isoproterenol-stimulated cAMP in transfected cells. Activation of adenylylcyclase by isoproterenol was inhibited ca. 30% by fMetLeuPhe in membranes prepared from transfected cells but not in membranes prepared from neutrophils. Prior treatment of transfected cells with pertussis toxin abrogated the inhibitory effect of fMetLeuPhe. These data indicate that factors in addition to the primary structure of the formylpeptide receptor govern its transductional activities.