Novel mechanism of plasma prekallikrein (PK) activation by vascular smooth muscle cells: evidence of the presence of PK activator.

The contribution of plasma prekallikrein (PK) to vascular remodeling is becoming increasingly recognized. Plasma PK is activated when the zymogen PK is digested to an active enzyme by activated factor XII (FXII). Here, we present our findings that vascular smooth muscle cells (VSMC) activate plasma PK in the absence of FXII. Extracted plasma membrane and cytosolic fractions of VSMCs activate PK, but the rate of PK activation was greater by the membrane fraction. FXII neutralizing antibody did not affect PK activation by extracted proteins of VSMCs. VSMC PKA was inhibited by the serine protease inhibitors such as aprotinin, phenylmethylsulfonyl fluoride, leupeptin and CTI with CI50 of 0.78 µM, 1 mM, 3.13 µM and 40 nM on the cultured cells, respectively. No inhibition of PK activation by cysteine, aspartic acid, and metalloprotease inhibitors was observed. This is the first report of the presence of an intrinsic PKA in VSMC. Considering that VSMCs are normally separated from the circulating blood by endothelial cells, direct PK activation by VSMCs may play a role in disease states like diabetes, hyperlipidemia or hypertension where the endothelial layer is damaged.

Regulation of tyrosine kinase cascades by G-protein-coupled receptors.

Mitogenic signaling by G-protein-coupled receptors (GPCRs) involves tyrosine phosphorylation of adaptor proteins and assembly of multiprotein Ras activation complexes. Over the past three years, three types of scaffolds for GPCR-directed complex assembly have been identified: transactivated receptor tyrosine kinases (RTKs), integrin-based focal adhesions, and GPCRs themselves. Nonreceptor tyrosine kinases play an important role in each case. The processes of GPCR desensitization and sequestration via clathrin-coated pits are also involved in signaling through the RTK- and GPCR-based scaffolds.

Antagonism of catecholamine receptor signaling by expression of cytoplasmic domains of the receptors.

The actions of many hormones and neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins). These receptors are characterized by a highly conserved topographical arrangement in which seven transmembrane domains are connected by intracellular and extracellular loops. The interaction between these receptors and G proteins is mediated in large part by the third intracellular loop of the receptor. Coexpression of the third intracellular loop of the alpha 1B-adrenergic receptor with its parent receptor inhibited receptor-mediated activation of phospholipase C. The inhibition extended to the closely related alpha 1C-adrenergic receptor subtype, but not the phospholipase C-coupled M1 muscarinic acetylcholine receptor nor the adenylate cyclase-coupled D1A dopamine receptor. These results suggest that the receptor-G protein interface may represent a target for receptor antagonist drugs.

Targeting the receptor-Gq interface to inhibit in vivo pressure overload myocardial hypertrophy.

Hormones and neurotransmitters may mediate common responses through receptors that couple to the same class of heterotrimeric guanine nucleotide-binding (G) protein. For example, several receptors that couple to Gq class proteins can induce cardiomyocyte hypertrophy. Class-specific inhibition of Gq-mediated signaling was produced in the hearts of transgenic mice by targeted expression of a carboxyl-terminal peptide of the alpha subunit Galphaq. When pressure overload was surgically induced, the transgenic mice developed significantly less ventricular hypertrophy than control animals. The data demonstrate the role of myocardial Gq in the initiation of myocardial hypertrophy and indicate a possible strategy for preventing pathophysiological signaling by simultaneously blocking multiple receptors coupled to Gq.

Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes.

The Ras-dependent activation of mitogen-activated protein (MAP) kinase pathways by many receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) requires the activation of Src family tyrosine kinases. Stimulation of beta2 adrenergic receptors resulted in the assembly of a protein complex containing activated c-Src and the receptor. Src recruitment was mediated by beta-arrestin, which functions as an adapter protein, binding both c-Src and the agonist-occupied receptor. beta-Arrestin 1 mutants, impaired either in c-Src binding or in the ability to target receptors to clathrin-coated pits, acted as dominant negative inhibitors of beta2 adrenergic receptor-mediated activation of the MAP kinases Erk1 and Erk2. These data suggest that beta-arrestin binding, which terminates receptor-G protein coupling, also initiates a second wave of signal transduction in which the "desensitized" receptor functions as a critical structural component of a mitogenic signaling complex.

Augmented mitogenic responsiveness to epidermal growth factor in murine fibroblasts that overexpress pp60c-src.

C3H10T1/2 murine fibroblasts overexpressing chicken pp60c-src showed a two- to fivefold enhanced incorporation of [3H]thymidine into DNA in response to epidermal growth factor (EGF) relative to that of the parent line. No difference in growth characteristics, number and affinity of EGF receptors, or hormone potency was attributable to c-src overexpression. These results suggest that pp60c-src may interact with the mitogenic signal transduction pathway of EGF in some event distal to hormone binding.

Platelet-derived growth factor receptor association with Na(+)/H(+) exchanger regulatory factor potentiates receptor activity.

Platelet-derived growth factor (PDGF) is a potent mitogen for many cell types. The PDGF receptor (PDGFR) is a receptor tyrosine kinase that mediates the mitogenic effects of PDGF by binding to and/or phosphorylating a variety of intracellular signaling proteins upon PDGF-induced receptor dimerization. We show here that the Na(+)/H(+) exchanger regulatory factor (NHERF; also known as EBP50), a protein not previously known to interact with the PDGFR, binds to the PDGFR carboxyl terminus (PDGFR-CT) with high affinity via a PDZ (PSD-95/Dlg/Z0-1 homology) domain-mediated interaction and potentiates PDGFR autophosphorylation and extracellular signal-regulated kinase (ERK) activation in cells. A point-mutated version of the PDGFR, with the terminal leucine changed to alanine (L1106A), cannot bind NHERF in vitro and is markedly impaired relative to the wild-type receptor with regard to PDGF-induced autophosphorylation and activation of ERK in cells. NHERF potentiation of PDGFR signaling depends on the capacity of NHERF to oligomerize. NHERF oligomerizes in vitro when bound with PDGFR-CT, and a truncated version of the first NHERF PDZ domain that can bind PDGFR-CT but which does not oligomerize reduces PDGFR tyrosine kinase activity when transiently overexpressed in cells. PDGFR activity in cells can also be regulated in a NHERF-dependent fashion by stimulation of the beta(2)-adrenergic receptor, a known cellular binding partner for NHERF. These findings reveal that NHERF can directly bind to the PDGFR and potentiate PDGFR activity, thus elucidating both a novel mechanism by which PDGFR activity can be regulated and a new cellular role for the PDZ domain-containing adapter protein NHERF.

Insulin and phorbol ester induce distinct phosphorylations of pp60c-src in the BC3H-1 murine myocyte cell line.

Insulin and phorbol esters have been shown to produce similar, non-additive metabolic effects in BC3H-1 murine myocytes. Recently, it has been demonstrated that insulin stimulation of these cells increases production of diacylglycerol, a known activator of protein kinase C (PK-C). To determine if insulin stimulation results in the activation of PK-C, we have examined the effects of insulin and the tumor promoting phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on the phosphorylation of a known PK-C substrate in vivo, the cellular proto-oncogene product, pp60c-src. Differentiated BC3H-1 monocytes showed an approximate twofold elevation in the [32P] content of pp60c-src following stimulation with insulin or TPA for 20 min, with no detectable change in the level of immunoprecipitable c-src protein. The enhanced phosphorylation in response to each agent localized to serine residues in the amino terminal 16 kD staphylococcal V8 proteolytic fragment. Tryptic phosphopeptide analysis revealed that TPA stimulation resulted in an approximate 18-fold increase in phosphorylation of the serine 12-containing tryptic fragment. Insulin stimulation, however, resulted in an approximate 10-fold increase in phosphorylation of the serine 17-containing tryptic fragment with little or no accompanying increase in serine 12 phosphorylation. In cells exposed to high concentrations of TPA for 16 h to deplete PK-C activity, insulin, but not TPA, stimulated phosphorylation of pp60c-src. These data suggest that insulin and phorbol ester induce phosphorylation of pp60c-src by distinct protein kinases.

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 beta-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.

'Location, location, location': activation and targeting of MAP kinases by G protein-coupled receptors.

A growing body of data supports the conclusion that G protein-coupled receptors can regulate cellular growth and differentiation by controlling the activity of MAP kinases. The activation of heterotrimeric G protein pools initiates a complex network of signals leading to MAP kinase activation that frequently involves cross-talk between G protein-coupled receptors and receptor tyrosine kinases or focal adhesions. The dominant mechanism of MAP kinase activation varies significantly between receptor and cell type. Moreover, the mechanism of MAP kinase activation has a substantial impact on MAP kinase function. Some signals lead to the targeting of activated MAP kinase to specific extranuclear locations, while others activate a MAP kinase pool that is free to translocate to the nucleus and contribute to a mitogenic response.

Behavioral retardation in a macaque with autosomal trisomy and aging mother.

The social development of a female rhesus monkey (Macaca mulatta) was followed from the day of birth until her death, at age 32 months. The subject, born to an older mother, had an extra autosome (karyotype: 43, XX, +18), an affliction that came about spontaneously. MRI scans revealed that she was also hydrocephalic. Compared to 23 female monkeys growing up under identical conditions, the subject showed serious motor deficiencies, a dramatic delay in the development of social behavior, poorly established dominance relationships, and greater than usual dependency on mother and kin. The subject was well-integrated into the social group, however.

Functional signaling biases in G protein-coupled receptors: Game Theory and receptor dynamics.

Pharmacotherapeutic targeting of G protein-coupled receptors (GPCRs) is perhaps the most important field of drug design, as agents designed to control these receptors constitute more than half of the pharmacopeia. Initially GPCRs were considered to be unitary entities, possessing all of their potential functionality in their characteristic heptahelical core. Early models of the functional activity of GPCRs considered them to possess just a simple 'on' or 'off' status. Recent research however has allowed us to realize that GPCR functionality is dependent upon many other proteins outside of the heptahelical core, on the site of GPCR expression in a tissue or a microdomain in a cell, and, most importantly, on the formation of differential 'active' states preferentially coupled to specific signal transduction structures. The recognition of such signaling diversity has facilitated the ability to appreciate and identify ligands for GPCRs that demonstrate a bias towards one signaling form of a receptor to another. However while potentially increasing our ability for selective signal targeting, our approach to understanding the physiological ramifications of systemic signaling manipulation is underdeveloped. This explosion in the complexity of GPCR signaling is now becoming familiar territory to receptor biologists, yet the application of this knowledge to drug design is relatively limited. This review will attempt to outline potential pitfalls and unseen benefits of using signaling bias in therapeutic design as well as highlighting new applications such as Game Theory for uncovering new therapeutic applications for biased agonists.

Hexose-independent activation of glycogen synthase and pyruvate dehydrogenase by insulin is dissociated in the mouse BC3H-1 cell line.

We have studied the effects of insulin on several aspects of cell metabolism in the insulin-sensitive, nonfusing muscle cell line BC3H-1. In the absence of exogenous hexose, insulin did not alter basal glycogen synthase percentage I activity, or attenuate the increase in intracellular cAMP content, the activation of glycogen phosphorylase a, or the decrease in glycogen synthase I brought about by beta-adrenergic receptor activation with epinephrine. In contrast, both insulin and the tumor-promoting phorbol ester, tetradecanoylyl phorbol acetate markedly increased mitochondrial pyruvate dehydrogenase activity in the absence of hexose. Both glycogen synthase phosphatase and glycogen synthase kinase activities were present in BC3H-1 cell extracts and were regulated in the expected manner by glucose 6-phosphate and cAMP, respectively. Since the pattern of partial insulin resistance seen in BC3H-1 myocytes would require that several potentially insulin-sensitive enzymes be insensitive to insulin-generated signals, the most likely explanation for these data is that the myocytes are defective in some mechanism of insulin signaling which is independent of the mechanism for pyruvate dehydrogenase activation.

The similarity principle underlying social bonding among female rhesus monkeys.

Twenty adult female rhesus monkeys (Macaca mulatta) were observed over a three-year period. They lived in a mixed captive group with kinship relations known for three generations. The study's aim was to test Seyfarth's [J. theor. Biol. 65: 671-698, 1977] model of rank-related grooming and to investigate two other possible determinants of social bonding, i.e. relative age and the group's stratification into two social classes. Data on affiliation, coalitions, and social competition were collected by means of both focal observation and instantaneous time sampling. Whereas certain elements of the existing model were confirmed, its explanatory principles were not. Social competition did not result in more contact among close-ranking females (the opposite effect was found), and the relation between affiliative behavior and coalitions was more complex than predicted. Based on multivariate analyses and a comparison of theoretical models, we propose a simpler, more encompassing principle underlying interfemale attraction. According to this 'similarity principle', rhesus females establish bonds with females whom they most resemble. The similarity may concern genetical and social background, age, hierarchical position and social class. Effects of these four factors were independently demonstrated. The most successful model assumed that similarity factors influence female bonding in a cumulative fashion.

G protein-coupled receptors desensitize and down-regulate epidermal growth factor receptors in renal mesangial cells.

Different types of plasma membrane receptors engage in various forms of cross-talk. We used cultures of rat renal mesangial cells to study the regulation of EGF receptors (EGFRs) by various endogenous G protein-coupled receptors (GPCRs). GPCRs (5-hydroxytryptamine(2A), lysophosphatidic acid, angiotensin AT(1), bradykinin B(2)) were shown to transactivate EGFRs through a protein kinase C-dependent pathway. This transactivation resulted in the initiation of multiple cellular signals (phosphorylation of the EGFRs and ERK and activation of cAMP-responsive element-binding protein (CREB), NF-kappaB, and E2F), as well as subsequent rapid down-regulation of cell-surface EGFRs and internalization and desensitization of the EGFRs without change in the total cellular complement of EGFRs. Internalization of the EGFRs and the down-regulation of cell-surface receptors in mesangial cells were blocked by pharmacological inhibitors of clathrin-mediated endocytosis and in HEK293 cells by transfection of cDNA constructs that encode dominant negative beta-arrestin-1 or dynamin. Whereas all of the effects of GPCRs on EGFRs were dependent to a great extent on protein kinase C, those initiated by EGF were not. These studies demonstrate that GPCRs can induce multiple signals through protein kinase C-dependent transactivation of EGFRs. Moreover, GPCRs induce profound desensitization of EGFRs by a process associated with the loss of cell-surface EGFRs through clathrin-mediated endocytosis.

Mitogenic signaling in androgen sensitive and insensitive prostate cancer cell lines.

PURPOSE: To investigate the role of a specific mitogen activated protein kinase, extracellular signal-regulated kinase (ERK), in regulating cell proliferation induced by three potentially important prostate cancer mitogens that signal via different classes of receptors. MATERIALS AND METHODS: Androgen sensitive (LNCaP) and insensitive (PC-3) prostate cancer cell lines were used in these studies. Epidermal growth factor (EGF), lysophosphatidic acid (LPA), and dihydrotestosterone (DHT) were the mitogenic stimulants and AG1478, a receptor tyrosine kinase inhibitor, and PD98059, an inhibitor of MEK, were the chemical inhibitors used in this study. Cell proliferation was measured using the WST-1 assay and ERK expression and activation was determined by immunoblotting for phospho- and total ERK. RESULTS: In androgen-sensitive LNCaP cells, epidermal growth factor (EGF) and dihydrotestosterone (DHT) both enhanced cell proliferation. EGF-stimulation dramatically increased ERK phosphorylation while DHT did not. In the androgen-insensitive cell line, PC-3, EGF- and LPA-induced ERK phosphorylation and cell proliferation. Inhibition of EGF- and LPA- induced ERK activation with the EGF receptor inhibitor, AG1478, or the MEK inhibitor, PD98059, attenuated their proliferative effects. Neither inhibitor had an effect on DHT stimulated cell proliferation. CONCLUSIONS: These data demonstrate heterogeneity of mitogenic signaling in prostate cancer cells, and support the hypothesis that androgens and growth factors utilize divergent signaling pathways in prostate cancer to induce proliferation.

Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A.

Many of the G-protein-coupled receptors for hormones that bind to the cell surface can signal to the interior of the cell through several different classes of G protein. For example, although most of the actions of the prototype beta2-adrenergic receptor are mediated through Gs proteins and the cyclic-AMP-dependent protein kinase (PKA) system, beta-adrenergic receptors can also couple to Gi proteins. Here we investigate the mechanism that controls the specificity of this coupling. We show that in HEK293 cells, stimulation of mitogen-activated protein (MAP) kinase by the beta2-adrenergic receptor is mediated by the betagamma subunits of pertussis-toxin-sensitive G proteins through a pathway involving the non-receptor tyrosine kinase c-Src and the G protein Ras. Activation of this pathway by the beta2-adrenergic receptor requires that the receptor be phosphorylated by PKA because it is blocked by H-89, an inhibitor of PKA. Additionally, a mutant of the receptor, which lacks the sites normally phosphorylated by PKA, can activate adenylyl cyclase, the enzyme that generates cAMP, but not MAP kinase. Our results demonstrate that a mechanism previously shown to mediate uncoupling of the beta2-adrenergic receptor from Gs and thus heterologous desensitization (PKA-mediated receptor phosphorylation), also serves to 'switch' coupling of this receptor from Gs to Gi and initiate a new set of signalling events.

Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attenuates G beta gamma-mediated signaling.

The beta gamma subunits (G beta gamma) of heterotrimeric G proteins modulate the activity of several signal-transducing effector molecules including G protein-coupled receptor kinases. G beta gamma binds to the carboxyl terminus of the beta-adrenergic receptor kinase (beta ARK) and regulates its activity. To investigate the effect of such a G beta gamma-binding domain on heterologous G beta gamma interactions, various receptors that can stimulate phospholipase C and/or type II adenylate cyclase were coexpressed in COS-7 cells with the carboxyl terminus of beta ARK1. Phosphoinositol hydrolysis in response to activation of receptors that stimulate phospholipase C via Gi beta gamma (alpha 2-adrenergic and M2-muscarinic cholinergic receptors) was markedly inhibited by the coexpressed beta ARK1 polypeptide, whereas that mediated by Gq alpha subunits (alpha 1-adrenergic and M1-muscarinic cholinergic receptors) was unaffected. Increased cellular cAMP levels due to stimulation of receptors and coexpressed adenylate cyclase II displayed marked inhibition in the presence of the beta ARK1 polypeptide. Moreover, inhibition of adenylate cyclase produced by alpha 2-adrenergic receptor stimulation (a Gi alpha-mediated process) was unaffected, indicating that the beta ARK1 polypeptide provides a useful tool for distinguishing between G alpha and G beta gamma pathways.