Polarization of chemoattractant receptor signaling during neutrophil chemotaxis.

Morphologic polarity is necessary for chemotaxis of mammalian cells. As a probe of intracellular signals responsible for this asymmetry, the pleckstrin homology domain of the AKT protein kinase (or protein kinase B), tagged with the green fluorescent protein (PHAKT-GFP), was expressed in neutrophils. Upon exposure of cells to chemoattractant, PHAKT-GFP is recruited selectively to membrane at the cell's leading edge, indicating an internal signaling gradient that is much steeper than that of the chemoattractant. Translocation of PHAKT-GFP is inhibited by toxin-B from Clostridium difficile, indicating that it requires activity of one or more Rho guanosine triphosphatases.

Gbetagamma and palmitate target newly synthesized Galphaz to the plasma membrane.

The subcellular location of a signaling protein determines its ability to transmit messages accurately and efficiently. Three different lipid modifications tether heterotrimeric G proteins to membranes: alpha subunits are myristoylated and/or palmitoylated, and gamma subunits are prenylated. In a previous study, we examined the role of lipid modifications in maintaining the membrane attachment of a G protein alpha subunit, alphaz, which is myristoylated and palmitoylated (Morales, J., Fishburn, C. S., Wilson, P. T., and Bourne, H. R. (1998) Mol. Biol. Cell 9, 1-14). Now we extend this analysis by characterizing the mechanisms that target newly synthesized alphaz to the plasma membrane (PM) and analyze the role of lipid modifications in this process. In comparison with newly synthesized alphas, which is palmitoylated but not myristoylated, alphaz moves more rapidly to the membrane fraction following synthesis in the cytosol. Newly synthesized alphaz associates randomly with cellular membranes, but with time accumulates at the PM. Palmitoylated alphaz is present only in PM-enriched fractions, whereas a nonpalmitoylated mutant of alphaz (alphazC3A) associates less stably with the PM than does wild-type alphaz. Expression of a C-terminal fragment of the beta-adrenoreceptor kinase, which sequesters free betagamma, impairs association of both alphaz and alphazC3A with the PM, suggesting that the alpha subunit must bind betagamma in order to localize at the PM. Based on these findings, we propose a model in which, following synthesis on soluble ribosomes, myristoylated alphaz associates randomly and reversibly with membranes; upon association with the PM, alphaz binds betagamma, which promotes its palmitoylation, thus securing it in the proper place for transmitting the hormonal signal.

C5a receptor activation. Genetic identification of critical residues in four transmembrane helices.

Hormones and sensory stimuli activate serpentine receptors, transmembrane switches that relay signals to heterotrimeric guanine nucleotide-binding proteins (G proteins). To understand the switch mechanism, we subjected 93 amino acids in transmembrane helices III, V, VI, and VII of the human chemoattractant C5a receptor to random saturation mutagenesis. A yeast selection identified 121 functioning mutant receptors, containing a total of 523 amino acid substitutions. Conserved hydrophobic residues are located on helix surfaces that face other helices in a modeled seven-helix bundle (Baldwin, J. M., Schertler, G. F., and Unger, V. M. (1997) J. Mol. Biol. 272, 144-164), whereas surfaces predicted to contact the surrounding lipid tolerate many substitutions. Our analysis identified 25 amino acid positions resistant to nonconservative substitutions. These appear to comprise two distinct components of the receptor switch, a surface at or near the extracellular membrane interface and a core cluster in the cytoplasmic half of the bundle. Twenty-one of the 121 mutant receptors exhibit constitutive activity. Amino acids substitutions in these activated receptors predominate in helices III and VI; other activating mutations truncate the receptor near the extracellular end of helix VI. These results identify key elements of a general mechanism for the serpentine receptor switch.

Receptor and betagamma binding sites in the alpha subunit of the retinal G protein transducin.

Transmembrane receptors for hormones, neurotransmitters, light, and odorants mediate their cellular effects by activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Crystal structures have revealed contact surfaces between G protein subunits, but not the surfaces or molecular mechanism through which Galphabetagamma responds to activation by transmembrane receptors. Such a surface was identified from the results of testing 100 mutant alpha subunits of the retinal G protein transducin for their ability to interact with rhodopsin. Sites at which alanine substitutions impaired this interaction mapped to two distinct Galpha surfaces: a betagamma-binding surface and a putative receptor-interacting surface. On the basis of these results a mechanism for receptor-catalyzed exchange of guanosine diphosphate for guanosine triphosphate is proposed.

Carboxyl-terminal mutations of Gq alpha and Gs alpha that alter the fidelity of receptor activation.

The carboxyl terminus of the G protein alpha subunit is a key determinant of the fidelity of receptor activation. We have previously shown that the Gq alpha subunit (alpha q) can be made to respond to alpha i-coupled receptors by replacing its carboxyl terminus with the corresponding alpha i2, alpha o, alpha z residues. We now extend these findings in three ways: 1) carboxyl-terminal mutations of alpha q/alpha i chimeras show that the critical amino acids are in the -3 and -4 positions, 2) exchange of carboxyl termini between alpha q and alpha z allows activation by receptors appropriate to the carboxyl-terminal residues, and 3) we identify receptors that either do or do not activate the expected carboxyl-terminal chimeras (alpha q/alpha i, alpha q/alpha s, alpha s/alpha q). Replacement of the five carboxyl-terminal amino acids of alpha q with the alpha s sequence permitted an alpha s-coupled receptor (the V2 vasopressin receptor but not the beta 2-adrenergic receptor) to stimulate phospholipase C. Replacement of the five carboxyl-terminal amino acids of alpha z with residues of alpha q permitted certain alpha q-coupled receptors (bombesin and V1a vasopressin receptors but not the oxytocin receptor) to stimulate adenylyl cyclase. Thus, the relative importance of the G alpha carboxyl terminus in permitting coupling to a new receptor depends on the receptor with which it is paired. These studies refine our understanding and provide new tools with which to study the fidelity of receptor/G alpha activation.

Rapid GDP release from Gs alpha in patients with gain and loss of endocrine function.

Luteinizing hormone stimulates testicular Leydig cells to produce testosterone by binding to a receptor that activates the G protein Gs and adenylyl cyclase. Testotoxicosis is a form of precocious puberty in which the Leydig cells secrete testosterone in the absence of luteinizing hormone, often due to constitutive activation of the luteinizing hormone receptor and (indirectly) Gs (refs 1-4). Here we study two unrelated boys suffering from a paradoxical combination of testotoxicosis and pseudohypoparathyroidism type Ia (PHP-Ia), a condition marked by resistance to hormones acting through cyclic AMP (parathyroid hormone and thyroid-stimulating hormone) as well as a 50% decrease in erythrocyte Gs activity (the remaining 50% is due to the normal Gs allele). In both patients, a mutation in the gene encoding the Gs alpha-subunit replace alanine at position 366 with serine. We show that this alpha s-A366S mutation constitutively activates adenylyl cyclase in vitro, causing hormone-independent cAMP accumulation when expressed in cultured cells, and accounting for the testotoxicosis phenotype (as cAMP stimulates testosterone secretion). Although alpha s-A366S is quite stable at testis temperature, it is rapidly degraded at 37 degrees C explaining the PHP-Ia phenotype caused by loss of Gs activity. In vitro experiments indicate that accelerated release of GDP causes both the constitutive activity and the thermolability of alpha s-A366S.

Type II adenylylcyclase integrates coincident signals from Gs, Gi, and Gq.

Agonists for Gi-coupled receptors augment Gs-stimulated cAMP synthesis in human embryonic kidney (HEK) 293 cells transiently expressing the type II isozyme of adenylylcyclase (AC-II). This augmentation, mediated by beta gamma subunits released from activated Gi, can be blocked by expression of the alpha subunit (alpha t) of retinal transducin (Gt), which presumably sequesters free beta gamma (Federman, A. D., Conklin, B. R., Schrader, K. A., Reed, R. R., and Bourne, H. R. (1992) Nature 356, 159-161). The alpha subunit of Gq, representing a G protein family distinct from both Gs and Gi, mimicked the inhibitory effect of alpha t, suggesting that hormonal stimulation of endogenous Gq might also release beta gamma subunits and thereby augment AC-II activity. Agonists for either of two Gq-coupled receptors did augment Gs-stimulated cAMP synthesis in HEK-293 cells expressing AC-II, but this effect was not blocked by expression of alpha t. The increased stimulation of AC-II was probably not mediated by the release of beta gamma subunits from Gq but rather by activation of protein kinase C (PKC) because of the following. (a) Phorbol esters, which activate PKC directly, elevated cAMP 2-fold in HEK-293 cells transfected with AC-II; this increase was synergistic with Gs-mediated activation of AC-II. (b) Treatments that partially inhibit or down-regulate PKC also partially prevented stimulation of AC-II by phorbol esters or by agonists for Gq-coupled receptors. Taken together, these results indicate that AC-II can integrate regulatory signals transmitted by at least three classes of G proteins; extracellular signals acting through Gs are enhanced synergistically by simultaneous signals transduced by Gi or Gq and mediated via beta gamma or PKC, respectively.