Gutkind Js

The Pathways Connecting G Protein-coupled Receptors to the Nucleus through Divergent Mitogen-activated Protein Kinase Cascades

Receptors coupled to heterotrimeric GTP-binding proteins (G proteins) are integral membrane proteins involved in the transmission of signals from the extracellular environment to the cytoplasm. The best known family of G protein-coupled receptors (GPCRs), currently comprising more than 1000 members, exhibits a common structural motif consisting of seven membrane-spanning regions (1) (Fig. 1). A diverse array of external stimuli including neurotransmitters, hormones, phospholipids, photons, odorants, certain taste ligands, and growth factors can activate specific members of this receptor family and promote interaction between the receptor and the G protein on the intracellular side of the membrane. This causes the exchange of GDP for GTP bound to the G protein a subunit and apparently the dissociation of the bg heterodimers. In turn, GTP-bound G protein a subunits or bg complexes initiate intracellular signaling responses by acting on effector molecules such as adenylate cyclases or phospholipases or directly regulating ion channel or kinase function (Fig. 1, and see below). Sixteen distinct mammalian G protein a subunits have been molecularly cloned and are divided into four families based upon sequence similarity: as, ai, aq, and a12. Similarly, eleven G protein g subunits and five G protein b subunits have been identified. Thus, GPCRs are likely to represent the most diverse signal transduction systems in eukaryotic cells. The biochemical and biological consequences of such diversity in subunit composition and coupling specificity for each receptor have just begun to be elucidated. In this review, we will briefly describe the role of G proteins and their coupled receptors in normal growth control and tumorigenesis and then focus on current efforts to elucidate the signaling pathways connecting this class of cell surface receptors to nuclear events regulating gene expression.

A Novel PDZ Domain Containing Guanine Nucleotide Exchange Factor Links Heterotrimeric G Proteins to Rho

Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. Here, we used a consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) to search DNA data bases, and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF that was termed Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activated biological and biochemical pathways specific for Rho, and activation of these pathways required an intact DH and PH domain. However, the PDZ domain was dispensable for these functions, and mutants lacking the LH domain were more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain revealed a limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This prompted us to investigate whether PDZ-RhoGEF could interact with representative members of each G protein family. We found that PDZ-RhoGEF was able to form, in vivo, stable complexes with two members of the Gα12 family, Gα12 and Gα13, and that this interaction was mediated by the LH domain. Furthermore, we obtained evidence to suggest that PDZ-RhoGEF mediates the activation of Rho by Gα12 and Gα13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of members of the Gα12 family which, in turn, activate an exchange factor acting on Rho.

Activation of Akt/Protein Kinase B by G Protein-coupled Receptors A ROLE FOR α AND βγ SUBUNITS OF HETEROTRIMERIC G PROTEINS ACTING THROUGH PHOSPHATIDYLINOSITOL-3-OH KINASEγ

The serine/threonine protein kinase Akt has recently been shown to be implicated in the pathway leading to cell survival in response to serum and growth factors in a variety of cellular systems. However, the existence of a biochemical route connecting this kinase to the large family of receptors that signal through heterotrimeric G proteins is yet to be explored. In this study, we set out to investigate whether GTP-binding protein (G protein)-coupled receptors (GPCRs) can stimulate Akt activity and survival pathways and, if so, to define the mechanism(s) whereby this class of cell surface receptors could regulate Akt function. Using ectopic expression of GPCRs in COS-7 cells as a model, we have observed that both m1 and m2 muscarinic acetylcholine receptors, representative of those GPCRs coupled to Gq and Giproteins, respectively, can readily activate an epitope-tagged form of Akt kinase and prevent UV-induced apoptosis. We have also found that the pathway connecting G proteins to Akt implicates signals emanating from Gαq, Gαi, and βγ dimers, but not from Gαs or Gα12, in each case acting through a pathway that involves a phosphatidylinositol-3-OH kinase activity. Moreover, our findings suggest a role for a novel βγ-sensitive complex, p101·phosphatidylinositol-3-OH kinase-γ, in the transduction of signals leading to Akt stimulation and cell survival by GPCRs and open new avenues for research on the function of the large family of G protein-linked receptors in the regulation of anti-apoptotic pathways.

Signaling from the Small GTP-binding Proteins Rac1 and Cdc42 to the c-Jun N-terminal Kinase/Stress-activated Protein Kinase Pathway A ROLE FOR MIXED LINEAGE KINASE 3/PROTEIN-TYROSINE KINASE 1, A NOVEL MEMBER OF THE MIXED LINEAGE KINASE FAMILY

Certain small GTP-binding proteins control the enzymatic activity of a family of closely related serine-threonine kinases known as mitogen-activated protein kinases (MAPKs). In turn, these MAPKs, such as p44mapk and p42mapk, referred to herein as MAPKs, and stress-activated protein kinases, also termed c-Jun N-terminal kinases (JNKs), phosphorylate and regulate the activity of key molecules that ultimately control the expression of genes essential for many cellular processes. Whereas Ras controls the activation of MAPK, we and others have recently observed that two members of the Rho family of small GTP-binding proteins, Rac1 and Cdc42, regulate the activity of JNKs. The identity of molecules communicating Rac1 and Cdc42 to JNK is still poorly understood. It has been suggested that Pak1 is the most upstream kinase connecting these GTPases to JNK; however, we have observed that coexpression of Pak1 with activated forms of Cdc42 or Rac1 diminishes rather than enhances JNK activation. This prompted us to explore the possibility that kinases other than Pak might participate in signaling from GTP-binding proteins to JNK. In this regard, a computer-assisted search for proteins containing areas of homology to that in Pak1 that is involved in binding to Rac1 and Cdc42 led to the identification of mixed lineage kinase 3 (MLK3), also known as protein-tyrosine kinase 1, as a potential candidate for this function. In this study, we found that MLK3 overexpression is sufficient to activate JNK potently without affecting the phosphorylating activity of MAPK or p38. Furthermore, we present evidence that MLK3 binds the GTP-binding proteins Cdc42 and Rac1 in vivo and that MLK3 mediates activation of MEKK-SEK-JNK kinase cascade by Rac1 and Cdc42. Taken together, these findings strongly suggest that members of the novel MLK family of highly related kinases link small GTP-binding proteins to the JNK signaling pathway.

SELECTIVE ACTIVATION OF EFFECTOR PATHWAYS BY BRAIN-SPECIFIC G PROTEIN BETA5

While multiple G protein β and γ subunit isoforms have been identified, the implications of this potential diversity of βγ heterodimers for signaling through βγ-regulated effector pathways remains unclear. Furthermore the molecular mechanism(s) by which the βγ complex modulates diverse mammalian effector molecules is unknown. Effector signaling by the structurally distinct brain-specific β5 subunit was assessed by transient cotransfection with γ2 in COS cells and compared with β1. Transfection of either β1 or β5 with γ2 stimulated the activity of cotransfected phospholipase C-β2 (PLC-β2), as previously reported. In contrast, cotransfection of β1 but not β5 with γ2 stimulated the mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways even though the expression of β5 in COS cells was evident by immunoblotting. The G protein β5 expressed in transfected COS cells was properly folded as its pattern of stable C-terminal proteolytic fragments was identical to that of native brain β5. The inability of β5 to activate the MAPK and JNK pathways was not overcome by cotransfection with three additional Gγ isoforms. These results suggest it is the Gβ subunit which determines the pattern of downstream signaling by the βγ complex and imply that the structural features of the βγ complex mediating effector regulation may differ among effectors.

Role of mitogen-activated protein kinases and c-Jun/AP-1 trans-activating activity in the regulation of protease mRNAs and the malignant phenotype in NIH 3T3 fibroblasts.

Ras activates a multitude of downstream activities with roles in cellular proliferation, invasion and metastasis, differentiation, and programmed cell death. In this work we have evaluated the requirement of extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase kinase (JNKK), and c-Jun/AP-1 activities in transformation and extracellular matrix invasion ofras oncogene expressing NIH 3T3 fibroblasts by expressing stable mutant genes that constitutively inhibit these activities. Whereas the inhibition of ERK activity reverts the transformed and invasive phenotype, the inhibition of the JNK pathway and AP-1trans-activating activities by JNKK[K129R] and c-Jun(TAM67) had no effect on the ability of the rasoncogene-expressing cells to grow in soft agar or invade Matrigel basement membrane. Thus an elevated JNK activity and/or c-Jun/AP-1trans-activating activity are not absolute requirements forras transformation or invasion through basement membrane, and the dependence on AP-1 activity for transformation is cell-specific. However, inhibition of JNK kinase (JNKK) inras-transformed cells with normally elevated JNK activity switches the protease-dependent invasive phenotype from a urokinase plasminogen activator (uPA)-dependent to a cathepsin L (CL)-dependent invasive phenotype. Conversely, treatment of ras-transformed cells of low constitutive JNK activity with the JNK stimulator, anisomycin, converts the protease mRNA levels from those characteristic of a CL-dependent to a uPA-dependent phenotype. These protease phenotypes can be duplicated in untransformed NIH 3T3 cells that express platelet-derived growth factor receptors and m1 muscarinic receptors that selectively stimulate the ERK or JNK pathways, respectively. It is concluded that high ERK activity is required for both protease phenotypes, whereas the JNK pathway and c-Jun/AP-1 activity are not required for transformation but regulate a switch between uPA and CL protease phenotypes in both transformed and untransformed cells. Inras-transformed NIH 3T3 fibroblasts, the uPA- and CL-dependent protease phenotypes are redundant in their ability to invade through basement membrane.

The Constitutively Active Mutant Gα13Transforms Mouse Fibroblast Cells Deficient in Insulin-like Growth Factor-I Receptor

Insulin-like growth factor-I (IGF-I) receptor plays an important role in normal cell cycle progression and tumor growth, and it is thought to be essential for cellular transformation. To test this hypothesis, we stably transfected a GTPase-deficient mutant human Gα13, which is highly oncogenic when overexpressed in vitro, into R− fibroblasts derived from IGF-I receptor-deficient mice. Northern blots of multiple clones revealed the expression of a 1.8-kilobase pair mutant Gα13 transcript in transfected cells, in addition to the 6-kilobase pair endogenous mRNA. The transfection resulted in a doubling of the expression of Gα13 protein in these cells as assessed by Western blot analysis. The transforming ability of the mutant Gα13 was tested using the soft agar assay. Nontransfected R− cells cultured with 10% fetal bovine serum failed to form colonies after 3 weeks. Most of the mutant Gα13-expressing clones formed significant numbers of colonies (11–50 colonies/1000 cells plated). Overexpression of the IGF-I receptor enabled R− cells to form colonies (27 colonies), and co-transfection of the mutant Gα13 caused a further increase in colony formation (117–153 colonies) in three of five clones analyzed. Apparently Gα13 works through pathways other than mitogen-activated protein kinase and c-Jun N-terminal kinase in transforming R− cells, because their activities were not significantly altered by the mutant Gα13 expression. These results demonstrate that Gα13 can induce cellular transformation through pathways apparently independent of the IGF-I receptor and that activation of the IGF-I receptor signaling pathways, although not essential for the transforming phenotype, enhances the effect of other pathways.

A C-terminal mutant of the G protein beta subunit deficient in the activation of phospholipase C-beta.

The molecular mechanism by which the G protein βγ complex modulates multiple mammalian effector pathways is unknown. Homolog-scanning mutagenesis of the G protein β subunit was employed to identify residues critical for the activation of phospholipase C-β2 (PLC-β2). A series of chimeras was made by introducing small segments of the Dictyostelium β subunit into a background of mammalian β1 and tested in COS cell cotransfection assays for their ability to activate PLC-β2 and assemble with mammalian γ2. A chimera that contained four Dictyostelium β substitutions within the C-terminal 14 residues was unable to activate PLC-β2 when cotransfected with γ, despite its demonstrable expression in a γ-dependent manner. Cotransfection of the mutant blocked m2 muscarinic receptor activation of PLC by a pertussis toxin-sensitive pathway. This C-terminal mutant retained the ability, however, to stimulate the mitogen-activated protein kinase pathway. These results imply that activation of different βγ-responsive effectors is mediated by distinct domains.