Role of mitogen-activated protein kinase kinase kinases in signal integration.

Mitogen-activated protein kinases (MAPKs) are members of a dynamic protein kinase network through which diverse stimuli regulate the spatio-temporal activities of complex biological systems. MAPKs regulate critical cellular functions required for homeostasis such as the expression of cytokines and proteases, cell cycle progression, cell adherence, motility and metabolism. MAPKs therefore influence cell proliferation, differentiation, survival, apoptosis and development. In vertebrates, five MAPK families are regulated by MAPK kinase kinase-MAPK kinase-MAPK (MKKK-MKK-MAPK) phosphorelay systems. There are at least 20 MKKKs that selectively phosphorylate and activate different combinations of the seven MKKs, resulting in a specific activation profile of members within the five MAPK families. MKKKs are differentially activated by upstream stimuli including cytokines, antigens, toxins and stress insults providing a mechanism to integrate the activation of different MAPKs with the cellular response to each stimulus. Thus, MKKKs can be considered as 'signaling hubs' that regulate the specificity of MAPK activation. In this review, we describe how the MKKK 'hub' function regulates the specificity of MAPK activation, highlighting MKKKs as targets for therapeutic intervention in cancer and other diseases.

Constitutive activation of G protein-coupled receptors as a result of selective substitution of a conserved leucine residue in transmembrane helix III.

Whereas numerous mutations of the human lutropin receptor (hLHR) and human TSH receptor (hTSHR) have been shown to cause constitutive activation of these receptors, it has been suggested that either the hFSHR as a whole, or the i3/TM VI region of the hFSHR, is less susceptible to mutation-induced constitutive activation. However, as shown herein, substitution of a highly conserved leucine residue in transmembrane III (TM III) of the hFSHR (Leu 111.18) with arginine causes a 5-fold increase in basal cAMP in transfected cells, consistent with a strong constitutive activation of the hFSHR. Interestingly, this mutant is unresponsive to further hormonal stimulation. Substitutions of hFSHR(L460) with lysine, alanine, or aspartate show that only arginine causes constitutive activation. However, all result in decreased FSH responsiveness, suggesting a role for L460 in FSH-stimulated cAMP production by the hFSHR. Because Leu 111.18 is highly conserved in rhodopsin-like G protein-coupled receptors (GPCRs), we tested the effects of substitution of the comparable leucine in the human beta2-adrenergic receptor (hbeta2-AR). Substitution of L124 in the hbeta2-AR with arginine, lysine, or alanine resulted in constitutive activation as evidenced by increased basal levels of cAMP that could be attenuated by an inverse agonist. In all cases, isoproterenol-stimulated cAMP was unaffected. Taken altogether, our data support a model whereby Leu 111.18 may play a general role in GPCRs by stabilizing them in an inactive state. Constitutive activation may arise by both a disruption of Leu 111.18 as well as the introduction of a specific residue that serves to stabilize the active state of the receptor.

Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation.

A 5-week-old male infant presented with severe bacterial infections and poor wound healing, suggesting a neutrophil defect. Neutrophils from this patient exhibited decreased chemotaxis, polarization, azurophilic granule secretion, and superoxide anion (O(2)(-)) production but had normal expression and up-regulation of CD11b. Rac2, which constitutes >96% of the Rac in neutrophils, is a member of the Rho family of GTPases that regulates the actin cytoskeleton and O(2)(-) production. Western blot analysis of lysates from patient neutrophils demonstrated decreased levels of Rac2 protein. Addition of recombinant Rac to extracts of the patient neutrophils reconstituted O(2)(-) production in an in vitro assay system. Molecular analysis identified a point mutation in one allele of the Rac2 gene resulting in the substitution of Asp57 by an Asn (Rac2(D57N)). Asp57 is invariant in all defined GTP-binding proteins. Rac2(D57N) binds GDP but not GTP and inhibits oxidase activation and O(2)(-) production in vitro. These data represent the description of an inhibitory mutation in a member of the Rho family of GTPases associated with a human immunodeficiency syndrome.

Certain activating mutations within helix 6 of the human luteinizing hormone receptor may be explained by alterations that allow transmembrane regions to activate Gs.

Male-limited gonadotropin-independent precocious puberty (MPP) is frequently associated with mutations of the human LH/CG receptor (hLHR) that result in constitutively active hLHRs. Many such activating mutations have been identified in transmembrane 6 of the hLHR, with the substitution of Asp-578 being the most frequently observed mutation. Mutagenesis of a transmembrane helix of a G protein-coupled receptor can cause local alterations in the conformation near the mutated residue, allosteric changes elsewhere in the protein, and/or changes in the interhelical packing of the receptor. Therefore, while it has been hypothesized that activation of the receptor by mutations of Asp-578 may arise via alterations in the interactions of helix 6 with other transmembrane helices and/or by allosterically altering the conformation of the third intracellular loop, it has not been possible to ascertain the role of the sixth transmembrane helix per se in activating Gs in the mutated full-length receptor. Recently, however, we have shown that a peptide KMAILIFT, corresponding to the juxtacytoplasmic portion of helix 6 of the hLHR, is capable of activating Gs. These results suggest that helix 6 itself can directly interact with Gs. Importantly, the KMAILIFT peptide did not include Asp-578, which lies just C-terminal to this sequence. We show herein that a peptide extended to include Asp-578 (KMAILIFTDFT) is a poor activator of Gs. However, if the peptide is synthesized with the aspartate replaced with either a glycine or tyrosine, substitutions that are found in some patients with MPP, these peptides have Gs-stimulating activity. Additionally, a transmembrane 6 peptide with the substitution of Ile-575 with leucine, another mutation found in MPP, mimicked the activating effects of this mutation in the full-length receptor. The ability of peptides in which Asp-578 or Ile-575 is substituted to mimic the activating effects of these mutations in the full-length receptor suggests that the sixth transmembrane helix represents a site for direct interaction with Gs. In addition to the stimulatory effects of transmembrane 6 peptides, peptides corresponding to the juxtacytoplasmic portions of the fourth, fifth, and seventh helices were also able to stimulate Gs. These results are consistent with the hypothesis that the transmembrane helices may form a pocket for interaction with Gs and that constitutive activation of the hLHR may involve the opening of the pocket formed by these helices, thus exposing Gs-binding sites on these helices.

Mutations that induce constitutive activation and mutations that impair signal transduction modulate the basal and/or agonist-stimulated internalization of the Lutropin/Choriogonadotropin receptor.

Previous results from this laboratory suggested that the same active conformation of the lutropin/choriogonadotropin receptor (LHR) is involved in the stimulation of G proteins and in triggering the internalization of the bound agonist. We have now analyzed two naturally occurring, constitutively active mutants of the human LHR. These mutations were introduced into the rat LHR (rLHR) and are designated L435R and D556Y. Cells expressing rLHR-D556Y bind human choriogonadotropin (hCG) with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation does not affect the internalization of the free receptor, but it enhances the internalization of the agonist-occupied receptors approximately 3-fold. Cells expressing rLHR-L435R also bind hCG with normal affinity, exhibit a 47-fold increase in basal cAMP, and do not respond to hCG with a further increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors approximately 2- and approximately 17-fold, respectively. We conclude that the state of activation of the rLHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing rLHR-L435R is due to the fast rate of internalization of the bound hCG. The finding that membranes expressing rLHR-L435R (a system where internalization does not occur) respond to hCG with an increase in adenylyl cyclase activity supports this suggestion.

A unique constitutively activating mutation in third transmembrane helix of luteinizing hormone receptor causes sporadic male gonadotropin-independent precocious puberty.

Several constitutively activating mutations have been demonstrated in the sixth transmembrane helix of the human LH receptor (hLHR) in boys with gonadotropin-independent precocious puberty. In the current study, we examined two unrelated Brazilian boys with gonadotropin-independent precocious puberty caused by two different heterozygous activating mutations of the hLHR. Direct sequencing of the entire exon 11 of the hLHR revealed a heterozygous substitution of T for G at nucleotide 1370, that converts Leu 457 to Arg in the third transmembrane helix of the hLHR in one affected boy. His biological parents had a normal hLHR gene sequence, establishing the sporadic nature of this novel Leu457Arg mutation. Human embryonic 293 cells expressing hLHR mutant (L457R) or hLHR wild-type bound CG with high affinity. However, cells expressing hLHR(L457R) exhibited significantly higher basal levels of cAMP (7- to 14-fold) than cells expressing the wild-type receptor, indicating constitutive activation of hLHR(L457R). Basal levels of cAMP in hLHR(L457R)-expressing cells were, nonetheless, not as great as the levels of cAMP produced by hLHR wild-type-expressing cells incubated with a saturating concentration of CG. Furthermore, cells expressing hLHR(L457R) were unresponsive to further stimulation by CG. This finding was confirmed in the patient by lack of an increase in serum testosterone after CG stimulation. These results suggest that the conformation of hLHR(L457R) mutant represents a different activated receptor state (R*) than the agonist-occupied wild-type receptor. We also identified the previously described Ala568Val mutation in the third intracellular loop of the LHR in the other affected African-Brazilian boy and his normal prepubertal sister, suggesting the inherited form of precocious puberty in this boy. We conclude that the third transmembrane helix is a potential area for activating mutations of the hLHR that cause male precocious puberty.

Evidence for the direct involvement of transmembrane region 6 of the lutropin/choriogonadotropin receptor in activating Gs.

The luteinizing hormone/chorionic gonadotropin receptor (LHR) is a heptahelical receptor that interacts primarily with Gs. Previous studies by others have shown that some forms of familial male precocious puberty are associated with mutations of the human LHR in the sixth transmembrane region that result in constitutive activation of the receptor. This study demonstrates that a peptide corresponding to the lower portion of the sixth transmembrane region of the LHR can significantly activate adenylyl cyclase activity. Experiments with membranes derived from wild-type versus cyc- S49 cells demonstrate that the stimulation of cyclase by this peptide is due to an activation of Gs. As such, our data demonstrate a direct role for transmembrane region 6 of the rat LHR in activating Gs and therefore raise the possibility that mutations in transmembrane region 6 of the LHR may directly affect the coupling of the receptor to Gs. Significantly, these data are the first to demonstrate the ability of a transmembrane portion of a G protein-coupled receptor, in the absence of any contributions from an intracellular loop region, to activate a G protein.