Constitutively active G protein-coupled receptor mutants block dictyostelium development

cAR1, a G protein-coupled receptor (GPCR) for cAMP, is required for the multicellular development of Dictyostelium. The activation of multiple pathways by cAR1 is transient because of poorly defined adaptation mechanisms. To investigate this, we used a genetic screen for impaired development to isolate four dominant-negative cAR1 mutants, designated DN1-4. The mutant receptors inhibit multiple cAR1-mediated responses known to undergo adaptation. Reduced in vitro adenylyl cyclase activation by GTPgammaS suggests that they cause constitutive adaptation of this and perhaps other pathways. In addition, the DN mutants are constitutively phosphorylated, which normally requires cAMP binding and possess cAMP affinities that are approximately 100-fold higher than that of wild-type cAR1. Two independent activating mutations, L100H and I104N, were identified. These residues occupy adjacent positions near the cytoplasmic end of the receptor's third transmembrane helix and correspond to the (E/D)RY motif of numerous mammalian GPCRs, which is believed to regulate their activation. Taken together, these findings suggest that the DN mutants are constitutively activated and block development by turning on natural adaptation mechanisms.     

The cyclic nucleotide specificity of three cAMP receptors in Dictyostelium.

cAMP receptors mediate signal transduction pathways during development in Dictyostelium. A cAMP receptor (cAR1) has been cloned and sequenced (Klein, P., Sun, T. J., Saxe, C. L., Kimmel, A. R., Johnson, R. L., and Devreotes, P. N. (1988) Science 241, 1467-1472) and recently several other cAR genes have been identified (Saxe, C. L., Johnson, R., Devreotes, P. N., and Kimmel, A. R. (1991a) Dev. Genet. 12, 6-13; Saxe, C. L., Johnson, R. L., Devreotes, P. N., and Kimmel, A. R. (1991b) Genes Dev. 5, 1-8). We have expressed three receptor subtypes, cAR1, cAR2, and cAR3, in growing cells and have investigated their affinity and pharmacological specificity in a series of [3H]cAMP binding studies. In phosphate buffer, there were two affinity states of about 30 and 300 nM for cAR1 and 20 and 500 nM for cAR3 but no detectable affinity for cAR2. In the presence of 3 M ammonium sulfate, there was one affinity state of 4 nM for cAR1 and 11 nM for cAR2 and two affinity states of approximately 4 and 200 nM for cAR3. The relative affinities of 14 cyclic nucleotide derivatives were tested for each cAR in ammonium sulfate. These studies suggest a model (Van Haastert, P. J. M., and Kien, E. (1983) J. Biol. Chem. 258, 9636-9642) in which cAMP binds to all three receptor subtypes by maintaining hydrogen bond interactions at the N6 and O3' positions. Interactions at the exocyclic oxygens of cAMP varied between the receptors; cAR2 and cAR3 lacked a stereoselective interaction at the axial oxygen which was present in cAR1. The cleft, which binds the adenine ring of cAMP, was hydrophobic in cAR1 and cAR3 but relatively polar in cAR2. The analog specificity of cAR1 and cAR3 in phosphate buffer was similar to that measured in ammonium sulfate though the derivatives' relative affinity to cAMP was reduced. We conclude that these cAMP receptor subtypes can be distinguished by distinct pharmacological properties which will allow selective activation of each cAR during development.     

Overexpression of the cAMP receptor 1 in growing Dictyostelium cells.

cAR1, the cAMP receptor expressed normally during the early aggregation stage of the Dictyostelium developmental program, has been expressed during the growth stage, when only low amounts of endogenous receptors are present. Transformants expressing cAR1 have 7-40 times over growth stage and 3-5-fold over aggregation stage levels of endogenous receptors. The high amounts of cAR1 protein expressed constitutively throughout early development did not drastically disrupt the developmental program; the onset of aggregation was delayed by 1-3 h, and then subsequent stages proceeded normally. The affinity of the expressed cAR1 was similar to that of the endogenous receptors in aggregation stage cells when measured either in phosphate buffer (two affinity states with Kd's of approximately 30 and 300 nM) or in 3 M ammonium sulfate (one affinity state with a Kd of 2-3 nM). When expressed during growth, cAR1 did not appear to couple to its normal effectors since these cells failed to carry out chemotaxis or to elevate cGMP or cAMP levels when stimulated with cAMP. However, cAMP stimulated phosphorylation, and loss of ligand binding of cAR1 did occur. Like aggregation stage control cells, the cAR1 protein shifted in apparent molecular mass from 40 to 43 kDa and became highly phosphorylated when exposed to cAMP. In addition, the number of surface cAMP binding sites in cAR1 cells was reduced by over 80% during prolonged cAMP stimulation. These results define a useful system to express altered cAR1 proteins and examine their regulatory functions.     

Zymogen activation in the streptokinase-plasminogen complex. Ile1 is required for the formation of a functional active site.

Plasminogen (Plgn) is usually activated by proteolysis of the Arg561-Val562 bond. The amino group of Val562 forms a salt-bridge with Asp740, which triggers a conformational change producing the active protease plasmin (Pm). In contrast, streptokinase (SK) binds to Plgn to produce an initial inactive complex (SK.Plgn) which subsequently rearranges to an active complex (SK.Plgn*) although the Arg561-Val562 bond remains intact. Therefore another residue must substitute for the amino group of Val562 and provide a counterion for Asp740 in this active complex. Two candidates for this counterion have been suggested: Ile1 of streptokinase and Lys698 of Plgn. We have investigated the reaction of SK mutants and variants of the protease domain of microplasminogen (muPlgn) in order to determine if either of these residues is the counterion. The mutation of Ile1 of SK decreases the activity of SK.Plgn* by 100-fold (Ile1Val) to >/= 104-fold (Ile1-->Ala, Gly, Trp or Lys). None of these mutations perturb the binding affinity of SK, which suggests that Ile1 is not required for formation of SK.Plgn but is necessary for SK.Plgn*. The substitution of Lys698 of muPlgn decreases the activity of SK.Plgn* by only 10-60-fold. In contrast with the Ile1 substitutions, the Lys698 mutations also decreased the dissociation constant of the SK complex by 15-50-fold. These observations suggest that Lys698 is involved in formation of the initial SK.Plgn complex. These results support the hypothesis that Ile1 provides the counterion for Asp740.     

The surface cyclic AMP receptors, cAR1, cAR2, and cAR3, promote Ca2+ influx in Dictyostelium discoideum by a G alpha 2-independent mechanism.

Activation of surface folate receptors or cyclic AMP (cAMP) receptor (cAR) 1 in Dictyostelium triggers within 5-10 s an influx of extracellular Ca2+ that continues for 20 s. To further characterize the receptor-mediated Ca2+ entry, we analyzed 45Ca2+ uptake in amoebas overexpressing cAR2 or cAR3, cARs present during multicellular development. Both receptors induced a cAMP-dependent Ca2+ uptake that had comparable kinetics, ion selectivity, and inhibitor profiles as folate- and cAR1-mediated Ca2+ uptake. Analysis of mutants indicated that receptor-induced Ca2+ entry does not require G protein alpha subunits G alpha 1, G alpha 2, G alpha 3, G alpha 4, G alpha 7, or G alpha 8. Overexpression of cAR1 or cAR3 in g alpha 2- cells did not restore certain G alpha 2-dependent events, such as aggregation, or cAMP-mediated activation of adenylate and guanylate cyclases, but these strains displayed a cAMP-mediated Ca2+ influx with kinetics comparable to wild-type aggregation-competent cells. These results suggest that a plasma membrane-associated Ca(2+)-influx system may be activated by at least four distinct chemoreceptors during Dictyostelium development and that the response may be independent of G proteins.     

Chemoattractant-mediated Rap1 activation requires GPCR/G proteins

Rap1 is rapidly activated upon chemoattractant stimulation and plays an important role in cell adhesion and cytoskeletal reorganization during chemotaxis. Here, we demonstrate that G-protein coupled receptors and G-proteins are essential for chemoattractant-mediated Rap1 activation in Dictyostelium. The rapid Rap1 activation upon cAMP chemoattractant stimulation was absent in cells lacking chemoattractant cAMP receptors cAR1/cAR3 or a subunit of the heterotrimeric G-protein complex Gα2. Loss of guanylyl cyclases GCA/SGC or a cGMP-binding protein GbpC exhibited no effect on Rap1 activation kinetics. These results suggest that Rap1, a key regulator for the regulation of cytoskeletal reorganization during cell movement, is activated through the G-protein coupled receptors cAR1/cAR3 and Gα2 proteins in a way independent of the cGMP signaling pathway.     

Arrestins function in cAR1 GPCR-mediated signaling and cAR1 internalization in the development of Dictyostelium discoideum

Oscillation of chemical signals is a common biological phenomenon, but its regulation is poorly understood. At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6-min intervals, directing cell migration. Although the G protein–coupled cAMP receptor cAR1 and ERK2 are both implicated in regulating the oscillation, the signaling circuit remains unknown. Here we report that D. discoideum arrestins regulate the frequency of cAMP oscillation and may link cAR1 signaling to oscillatory ERK2 activity. Cells lacking arrestins (adcB⁻C⁻) display cAMP oscillations during the aggregation stage that are twice as frequent as for wild- type cells. The adcB⁻C⁻ cells also have a shorter period of transient ERK2 activity and precociously reactivate ERK2 in response to cAMP stimulation. We show that arrestin domain–containing protein C (AdcC) associates with ERK2 and that activation of cAR1 promotes the transient membrane recruitment of AdcC and interaction with cAR1, indicating that arrestins function in cAR1-controlled periodic ERK2 activation and oscillatory cAMP signaling in the aggregation stage of D. discoideum development. In addition, ligand-induced cAR1 internalization is compromised in adcB⁻C⁻ cells, suggesting that arrestins are involved in elimination of high-affinity cAR1 receptors from cell surface after the aggregation stage of multicellular development.     

Genetic and biochemical evidence that trehalase is a substrate of cAMP-dependent protein kinase in yeast

In Saccharomyces cerevisiae, trehalase activity in crude extracts obtained from wild type cells was activated about 3-fold by preincubation with cAMP and ATP. The inactive trehalase fractionated by DEAE-Sephacel chromatography was activated by the addition of the cAMP-dependent protein kinase fraction from wild type cells in the presence of cAMP and ATP. Using the crude extract obtained from bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase, the stimulation of trehalase activity was observed in the absence of cAMP. The cAMP-dependent protein kinase of CYR3 mutant cells which had a high Ka value for cAMP in the phosphorylation reaction required a high cAMP concentration for activation of trehalase. Increased activation of partially purified inactive trehalase (Mr = 320,000) was observed to correlate with increased phosphorylation of a protein (Mr = 80,000) identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay results using various mutants altered in cAMP metabolism indicated that the activation and phosphorylation of inactive trehalase fractions depended on the cAMP concentration accumulated in mutant cells. Inactivation and dephosphorylation of active trehalase fractions were observed by treatment with alkaline phosphatase or crude cell extracts. The results indicated that the conversion of inactive form of trehalase to the active form is regulated by cAMP through cAMP-dependent protein kinase.     

Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium.

cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.     

The cell density factor CMF regulates the chemoattractant receptor cAR1 in Dictyostelium

Starving Dictyostelium cells aggregate by chemotaxis to cAMP when a secreted protein called conditioned medium factor (CMF) reaches a threshold concentration. Cells expressing CMF antisense mRNA fail to aggregate and do not transduce signals from the cAMP receptor. Signal transduction and aggregation are restored by adding recombinant CMF. We show here that two other cAMP-induced events, the formation of a slow dissociating form of the cAMP receptor and the loss of ligand binding, which is the first step of ligand-induced receptor sequestration, also require CMF. Vegetative cells have very few CMF and cAMP receptors, while starved cells possess approximately 40,000 receptors for CMF and cAMP. Transformants overexpressing the cAMP receptor gene cAR1 show a 10-fold increase of [3H]cAMP binding and a similar increase of [125I]CMF binding; disruption of the cAR1 gene abolishes both cAMP and CMF binding. In wild-type cells, downregulation of cAR1 with high levels of cAMP also downregulates CMF binding, and CMF similarly downregulates cAMP and CMF binding. This suggests that the cAMP binding and CMF binding are closely linked. Binding of approximately 200 molecules of CMF to starved cells affects the affinity of the majority of the cAR1 cAMP receptors within 2 min, indicating that an amplifying mechanism allows one activated CMF receptor to regulate many cARs. In cells lacking the G-protein beta subunit, cAMP induces a loss of cAMP binding, but not CMF binding, while CMF induces a reduction of CMF binding without affecting cAMP binding, suggesting that the linkage of the cell density-sensing CMF receptor and the chemoattractant cAMP receptor is through a G-protein.     

Follicle-stimulating hormone interacts with exoloop 3 of the receptor

The human follicle-stimulating hormone (FSH) receptor consists of two distinct domains of approximately 330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain including three exoloops and seven transmembrane helices. The exodomain binds the hormone with high affinity, and the resulting hormone/exodomain complex modulates the endodomain where receptor activation occurs. It has been an enigma whether the hormone interacts with the endodomain. In a step to address the question, exoloop 3 of (580)KVPLITVSKAK(590) was examined by Ala scan, multiple substitution, assays for hormone binding, cAMP and inositol phosphate (IP) induction, and photoaffinity labeling. We present the evidence for the interaction of FSH and exoloop 3. A peptide mimic of exoloop 3 specifically and saturably photoaffinity-labels FSH alpha but not FSH beta. This is in contrast to photoaffinity labeling of FSH beta by the peptide mimic of the N-terminal region of the receptor. Leu(583) and Ile(584) are crucial for the interaction of FSH and exoloop 3. Substitutions of these two residues enhanced the hormone binding affinity. This is due to the loss of the original side chains but not the introduction of new side chains. The Leu(583) and Ile(584) side chains appear to project in opposite directions. Ile(584) appears to be so specific and to require flexibility and stereo specificity so that no other amino acids can fit into its place. Leu(583) is less specific. The improvement in hormone binding by substitutions was offset by the severe impairment of signal generation of cAMP and/or inositol phosphate. For example, the Phe or Tyr substitution of Leu(583) improved the hormone binding and cAMP induction but impaired IP induction. On the other hand, the substitutions for Ile(584) and Lys(590) abolished the cAMP and IP induction. Our results open a logical question whether Leu(583), Ile(584), and Lys(590) interact with the exodomain and/or the hormone. The answers will provide new insights into the mechanisms of hormone binding and signal generation.     

Two cAMP receptors activate common signaling pathways in Dictyostelium.

Multiple signal transduction pathways within a single cell may share common components. In particular, seven different transmembrane helix receptors may activate identical pathways by interacting with the same G-proteins. Dictyostelium cells respond to cAMP using one such receptor, cAR1, coupled by a typical heterotrimeric G-protein to intracellular effectors. However, cells in which the gene for cAR1 has been deleted are unexpectedly still able to respond to cAMP. This implies either that certain responses are mediated by a different receptor than cAR1, or alternatively that a second, partially redundant receptor shares some of the functions of cAR1. We have examined the dose response and ligand specificity of one response, cAMP relay, and the dose response of another, cyclic GMP synthesis. In each case, the EC50 was approximately 100-fold higher and the maximal response was smaller in car1- than wild-type cells. These data indicate that cAR1 normally mediates responses to cAMP. The ligand specificity suggests that the responses seen in car1- mutants are mediated by a second receptor, cAR3. To test this hypothesis, we constructed a cell line containing deletions of both cAR1 and cAR3 genes. As predicted, these lines are totally insensitive to cAMP. We conclude that the functions of the cAR1 and cAR3 receptors are partially redundant and that both interact with the same heterotrimeric G-protein to mediate these and other responses.     

Galpha3 and protein kinase A represent cross-talking pathways for gene expression in Dictyostelium discoideum

Heterotrimeric G proteins and protein kinase A (PKA) are regulators of development in Dictyostelium discoideum. It has been reported that disruption of the Dictyostelium Galpha3 gene (galpha3-) blocks development and expression of several early development genes, characteristics that are reminiscent of mutants lacking the catalytic subunit of PKA (pkac-). The hypothesis that Galpha3 and PKA signaling pathways may interact to control developmental gene expression was tested by comparing the regulation of seven genes expressed early in development in the wild-type and in galpha3- and pkac- mutants, and comparing PKA activity in the wild-type and in a galpha3- mutant. The expression patterns of six genes were affected similarly by the Galpha3 and PKA mutations, while the expression of only one gene, the cAMP receptor 1 (cAR1), differed between the mutants. PKA activity, measured by phosphorylation of the PKA-specific substrate Kemptide, was higher in galpha3- cells than in wild-type cells, suggesting that Galpha3 normally exerts an inhibitory effect on PKA activity. Although some early development genes appear to require both Galpha3 and PKA for expression, the differing response of cAR1 expression and the inhibitory effect of Galpha3 on PKA activity suggest that Galpha3 and PKA are members of interacting pathways controlling gene expression early in development.     

Waterproofing the heme pocket. Role of proximal amino acid side chains in preventing hemin loss from myoglobin

The ability of myoglobin to bind oxygen reversibly depends critically on retention of the heme prosthetic group. Globin side chains at the Leu(89)(F4), His(97)(FG3), Ile(99)(FG5), and Leu(104)(G5) positions on the proximal side of the heme pocket strongly influence heme affinity. The roles of these amino acids in preventing heme loss have been examined by determining high resolution structures of 14 different mutants at these positions using x-ray crystallography. Leu(89) and His(97) are important surface amino acids that interact either sterically or electrostatically with the edges of the porphyrin ring. Ile(99) and Leu(104) are located in the interior region of the proximal pocket beneath ring C of the heme prosthetic group. The apolar amino acids Leu(89), Ile(99), and Leu(104) "waterproof" the heme pocket by forming a barrier to solvent penetration, minimizing the size of the proximal cavity, and maintaining a hydrophobic environment. Substitutions with smaller or polar side chains at these positions result in exposure of the heme to solvent, the appearance of crystallographically defined water molecules in or near the proximal pocket, and large increases in the rate of hemin loss. Thus, the naturally occurring amino acid side chains at these positions serve to prevent hydration of the His(93)-Fe(III) bond and are highly conserved in all known myoglobins and hemoglobins.     

Streptokinase triggers conformational activation of plasminogen through specific interactions of the amino-terminal sequence and stabilizes the active zymogen conformation

Cleavage of Arg(561)-Val(562) in plasminogen (Pg) generates plasmin (Pm) through a classical activation mechanism triggered by an insertion of the new amino terminus into a binding pocket in the Pg catalytic domain. Streptokinase (SK) circumvents this process and activates Pg through a unique nonproteolytic mechanism postulated to be initiated by the intrusion of Ile(1) of SK in place of Val(562). This hypothesis was evaluated in equilibrium binding and kinetic studies of Pg activation with an SK mutant lacking Ile(1) (SK(2--414)). SK(2--414) retained the affinity of native SK for fluorescein-labeled [Lys]Pg and [Lys]Pm but induced no detectable conformational activation of Pg. The activity of SK(2--414) was partially restored by the peptides SK(1--2), SK(1--5), SK(1--10), and SK(1--15), whereas Pg(562--569) peptides were much less effective. Active site-specific fluorescence labeling demonstrated directly that the active catalytic site was formed on the Pg zymogen by the combination of SK(1--10) and SK(2--414), whereas sequence-scrambled SK(1-10) was inactive. The characterization of SK(1--10) containing single Ala substitutions demonstrated the sequence specificity of the interaction. SK(1--10) did not restore activity to the further truncated mutant SK(55-414), which was correlated with the loss of binding affinity of SK(55--414) for labeled [Lys]Pm but not for [Lys]Pg. The studies support a mechanism for conformational activation in which the insertion of Ile(1) of SK into the Pg amino-terminal binding cleft occurs through sequence-specific interactions of the first 10 SK residues. This event and the preferentially higher affinity of SK(2--414) for the activated proteinase domain of Pm are thought to function cooperatively to trigger the conformational change and stabilize the active zymogen conformation.     

Pleiotropic effects of substitutions of a highly conserved leucine in transmembrane helix III of the human lutropin/choriogonadotropin receptor with respect to constitutive activation and hormone responsiveness

It has been shown previously that a naturally occurring mutation of the human LH/CG receptor (hLHR), which replaces L457 in helix III with arginine, results in a receptor that constitutively elevates basal cAMP but does not respond to human CG (hCG) with further cAMP production. In the present study, substitutions of L457 with several amino acids were examined. The constitutive activation of cAMP production was observed only when L457 was replaced with a positively charged residue. Although constitutive activation of the inositol phosphate pathway could not be detected when measuring inositol phosphate production, the use of a more sensitive reporter gene assay for protein kinase C activation revealed the constitutive activation of this pathway by the R- and K-substituted mutants. Therefore, L457 of the hLHR plays a key role in stabilizing the receptor in an inactive conformation. Molecular modeling shows that the insertion of R, K, or H at position 457 triggers the receptor transition toward an active state due to the proximity of an anionic amino acid, D578, in helix VI. These substitutions cause perturbations in helix III-helix VI and helix III-helix VII interactions that culminate in the opening of a solvent-accessible site in the cytosolic domains potentially involved in Gs recognition. Interestingly, L457R was completely unresponsive and the K- and H-substituted L457 hLHR mutants were significantly blunted in their cAMP responses to hCG stimulation. Cells expressing L457R were also unresponsive to hCG with regards to increased inositol phosphate production. Other substitutions of L457 were identified, though, that selectively permit the hormonal stimulation of only one of the two signaling pathways. These results suggest a pivotal role for L457 in hormone-stimulated signal transduction by the hLHR.     

The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates,a positive effect of caffeine and potential role of microtubules

To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2βγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2βγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2βγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2βγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2βγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2βγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing.