Kristen L. Pierce

Signalling: Seven-transmembrane receptors

Seven-transmembrane receptors, which constitute the largest, most ubiquitous and most versatile family of membrane receptors, are also the most common target of therapeutic drugs. Recent findings indicate that the classical models of G-protein coupling and activation of second-messenger-generating enzymes do not fully explain their remarkably diverse biological actions.

Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.

Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of β-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered β-arrestin-2 binding to the receptor and internalization of AT1aR-β-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-β-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, β-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged β-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with β-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with β-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to β-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in β-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to β-arrestin-2, and the association of β-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that β-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.

New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades.

Activation of classical second messenger cascades cannot fully explain the recently appreciated roles of heptahelical, or G-protein coupled receptors (GPCRs), in stimulation of mitogen activated protein kinase (MAPK) cascades. Rather, several distinct signaling mechanisms appear to contribute to GPCR-mediated MAPK activation. These include transactivation of the Epidermal Growth Factor Receptor (EGFR) via the autocrine/paracrine release of EGF-like ligands at the cell surface and scaffolding of MAPK cascades. A significant advance in the understanding of how GPCRs activate MAPK cascades is the discovery that β-arrestin, a protein well known for its roles in both receptor desensitization and internalization, serves as a scaffolding protein for at least two GPCR stimulated MAPK cascades, the extracellular signal regulated kinase (ERK) cascade and the c-jun N-terminal kinase 3 (JNK3) cascade. Together, these novel mechanisms of GPCR-mediated MAPK regulation may permit GPCRs in specific situations to control the temporal and spatial activity of MAPKs and thereby determine the consequences of GPCR stimulation with respect to transcriptional activation, cell proliferation and apoptosis.

Classical and new roles of |[beta]|-arrestins in the regulation of G-PROTEIN-COUPLED receptors

In the classical model of G-protein-coupled receptor (GPCR) regulation, arrestins terminate receptor signalling. After receptor activation, arrestins desensitize phosphorylated GPCRs, blocking further activation and initiating receptor internalization. This function of arrestins is exemplified by studies on the role of arrestins in the development of tolerance to, but not dependence on, morphine. Arrestins also link GPCRs to several signalling pathways, including activation of the non-receptor tyrosine kinase SRC and mitogen-activated protein kinase. In these cascades, arrestins function as adaptors and scaffolds, bringing sequentially acting kinases into proximity with each other and the receptor. The signalling roles of arrestins have been expanded even further with the discovery that the formation of stable receptor–arrestin complexes initiates photoreceptor apoptosis in Drosophila, leading to retinal degeneration. Here we review our current understanding of arrestin function, discussing both its classical and newly discovered roles.

Transactivation of the EGF receptor mediates IGF-1-stimulated shc phosphorylation and ERK1/2 activation in COS-7 cells.

The receptor for insulin-like growth factor 1 (IGF-1) mediates multiple cellular responses, including stimulation of both proliferative and anti-apoptotic pathways. We have examined the role of cross talk between the IGF-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR) in mediating responses to IGF-1. In COS-7 cells, IGF-1 stimulation causes tyrosine phosphorylation of the IGF-1R β subunit, the EGFR, insulin receptor substrate-1 (IRS-1), and the Shc adapter protein. Shc immunoprecipitates performed after IGF-1 stimulation contain coprecipitated EGFR, suggesting that IGF-1R activation induces the assembly of EGFR·Shc complexes. Tyrphostin AG1478, an inhibitor of the EGFR kinase, markedly attenuates IGF-1-stimulated phosphorylation of EGFR, Shc, and ERK1/2 but has no effect on phosphorylation of IGF-1R, IRS-1, and protein kinase B (Akt). Cross talk between IGF-1 and EGF receptors is mediated through an autocrine mechanism involving matrix metalloprotease-dependent release of heparin-binding EGF (HB-EGF), because IGF-1-mediated ERK activation is inhibited both by [Glu52]Diphtheria toxin, a specific inhibitor of HB-EGF, and the metalloprotease inhibitor 1,10-phenanthroline. These data demonstrate that IGF-1 stimulation of the IRS-1/PI3K/Akt pathway and the EGFR/Shc/ERK1/2 pathway occurs by distinct mechanisms and suggest that IGF-1-mediated “transactivation” of EGFR accounts for the majority of IGF-1-stimulated Shc phosphorylation and subsequent activation of the ERK cascade.

Cloning of a Carboxyl-terminal Isoform of the Prostanoid FP Receptor

An FP prostanoid receptor isoform, which appears to arise from alternative mRNA splicing, has been cloned from a mid-cycle ovine large cell corpus luteum library. The isoform, named the FPB receptor, is identical to the original isoform, the FPA, throughout the seven transmembrane domains, but diverges nine amino acids into the carboxyl terminus. In contrast to FPA, whose carboxyl terminus continues for another 46 amino acids beyond the nine shared residues, the FPB terminates after only one amino acid. The FPA isoform appears to arise by the failure to utilize a potential splice site, while a 3.2-kilobase pair intron is spliced out from the FP gene to generate the FPB isoform mRNA. The two isoforms have indistinguishable radioligand binding properties, but seem to differ in functional coupling to phosphatidylinositol hydrolysis. Thus, in COS-7 cells transiently transfected with either the FPA or the FPB receptor cDNAs, prostaglandin F2α stimulates inositol phosphate accumulation to the same absolute maximum, but the basal level of inositol phosphate accumulation is approximately 1.3-fold higher in cells transfected with the FPB as compared with cells transfected with the FPA isoform. Using the polymerase chain reaction, mRNA encoding the FPB isoform was identified in the ovine corpus luteum.

Activation of FP Prostanoid Receptor Isoforms Leads to Rho-mediated Changes in Cell Morphology and in the Cell Cytoskeleton

Prostaglandin F2α(PGF2α) exerts its biological effects by binding to and activating FP prostanoid receptors. These receptors, which include two isoforms, the FPA and FPB, have been cloned from a number of species and are members of the superfamily of G-protein-coupled receptors. Previous studies have shown that the activation of FP receptors leads to phosphatidylinositol hydrolysis, intracellular calcium release, and activation of protein kinase C. Here, we demonstrate that PGF2α treatment of 293-EBNA (Epstein-Barr nuclear antigen) cells that have been stably transfected with either the FPA or FPB receptor isoforms leads to changes in cell morphology and in the cell cytoskeleton. Specifically, cells treated with PGF2α show retraction of filopodia and become rounded, and actin stress fibers are formed. Pretreatment of the cells with bisindolylmaleimide I, a protein kinase C inhibitor, has no effect on the PGF2α-induced changes in cell morphology, although it does block the effects of phorbol myristate acetate on cell morphology. On the other hand, the PGF2α-induced changes in cell morphology and formation of actin stress fibers can be blocked by pretreatment of the cells with C3 exoenzyme, a specific inhibitor of the small G-protein, Rho. Consistent with FP receptor induced formation of actin stress fibers and focal adhesions, FPA receptor activation also leads to rapid (within two minutes) tyrosine phosphorylation of p125 focal adhesion kinase (FAK) which can be blocked by pretreating the cells with C3 exoenzyme. Taken together, these results suggest that the FP receptor isoforms are coupled to at least two second messenger pathways, one pathway associated with protein kinase C activation, and the other with activation of Rho.

Prostanoid receptor heterogeneity through alternative mRNA splicing.

Prostaglandin (PG) and thromboxane (TX) receptors are G-protein coupled receptors that mediate the physiological actions of the five principal prostanoid metabolites: PGD2, PGE2, PGF2alpha, PGI2 (prostacyclin) and TXA2. Five major subdivisions of the prostanoid receptor family have been defined pharmacologically which correspond to each of the metabolites as follows: DP, EP, FP IP and TP. The EP receptors have been further classified pharmacologically into the EP1, EP2, EP3 and EP4 subtypes. Molecular biological studies have resulted in the cloning of cDNAs encoding all of these prostanoid receptors. In addition, the cloning of these receptors has revealed further heterogeneity through the use of alternative mRNA splicing. Specifically, mRNA splice variants have been identified for the EP1, EP3, FP and TP receptors. Interestingly, except for the EP1 receptors, the mechanisms giving rise to these receptor isoforms involves the use of splice sites located in the cytoplasmic carboxyl termini of these receptors. Thus, the eight human EP3 isoforms that have been identified are otherwise identical except for their carboxyl termini. Similarly, the optional use of a potential splice site encoding the carboxyl terminus gives rise to each of the two FP and TP receptor isoforms. Because the carboxyl termini of G-protein coupled receptors are generally implicated in interactions with G-proteins, it is not surprising that these receptor isoforms differ mainly with respect to their activation of second messenger pathways and not in their pharmacological characteristics. Differences also exist with respect to their levels of constitutive activity (e.g., in the absence of agonist) and in their desensitization.

Replacement of the carboxylic acid group of prostaglandin F2α with a hydroxyl or methoxy substituent provides biologically unique compounds

Replacement of the carboxylic acid group of PGF2α with the non‐acidic substituents hydroxyl (‐OH) or methoxy (‐OCH3) resulted in an unexpected activity profile. Although PGF2α 1‐OH and PGF2α 1‐OCH3 exhibited potent contractile effects similar to 17‐phenyl PGF2α in the cat lung parenchymal preparation, they were approximately 1000 times less potent than 17‐phenyl PGF2α in stimulating recombinant feline and human FP receptors. In human dermal fibroblasts and Swiss 3T3 cells PGF2α 1‐OH and PGF2α 1‐OCH3 produced no Ca2+ signal until a 1 μM concentration was exceeded. Pretreatment of Swiss 3T3 cells with either 1 μM PGF2α 1‐OH or PGF2α 1‐OCH3 did not attenuate Ca2+ signal responses produced by PGF2α or fluprostenol. In the rat uterus, PGF2α 1‐OH was about two orders of magnitude less potent than 17‐phenyl PGF2α whereas PGF2α 1‐OCH3 produced only a minimal effect. Radioligand binding studies on cat lung parenchymal plasma membrane preparations suggested that the cat lung parenchyma does not contain a homogeneous population of receptors that equally respond to PGF2α1‐OH, PGF2α1‐OCH3, and classical FP receptor agonists. Studies on smooth muscle preparations and cells containing DP, EP1, EP2, EP3, EP4, IP, and TP receptors indicated that the activity of PGF2α 1‐OH and PGF2α 1‐OCH3 could not be ascribed to interaction with these receptors. The potent effects of PGF2α 1‐OH and PGF2α 1‐OCH3 on the cat lung parenchyma are difficult to describe in terms of interaction with the FP or any other known prostanoid receptor.

Delayed reversal of shape change in cells expressing FP(B) prostanoid receptors. Possible role of receptor resensitization.

Prostaglandin F2α(PGF2α) receptors are G-protein-coupled receptors consisting of two alternative mRNA splice variants, named FPA and FPB. As compared with the FPA isoform, the FPB isoform lacks the last 46 amino acids of the carboxyl terminus and, therefore, represents a truncated version of the FPA. We recently found (Pierce, K. L., Fujino, H., Srinivasan, D., and Regan, J. W. (1999)J. Biol. Chem. 274, 35944–35949) that stimulation of both isoforms with PGF2α leads to activation of a Rho signaling pathway, resulting in tyrosine phosphorylation of p125 focal adhesion kinase, formation of actin stress fibers, and cell rounding. Although the activation of Rho and subsequent cell rounding occur at a similar rate for both isoforms, we now report that following the removal of PGF2α the reversal of cell rounding is much slower for cells expressing the FPB isoform as compared with the FPA isoform. Thus, in HEK-293 cells that stably express the FPA isoform, the reversal of cell rounding appears to be complete after 1 h, whereas for FPB-expressing cells there is essentially no reversal even after 2 h. Similarly, the disappearance of stress fibers and dephosphorylation of p125 focal adhesion kinase following removal of agonist are much slower in FPB-expressing cells than in FPA-expressing cells. The mechanism of this differential reversal appears to involve a difference in receptor resensitization following the removal of agonist. Based upon whole cell radioligand binding, agonist-induced stimulation of inositol phosphate formation, and mobilization of intracellular Ca2+, the FPBisoform resensitizes more slowly than the FPA isoform. These findings suggest that the carboxyl terminus of the FPA is critical for resensitization and that the slower resensitization of the FPB isoform leads to prolonged signaling. This differential signaling distinguishes the FPA and FPB receptor isoforms and could be important toward understanding the physiological actions of PGF2α.