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.

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IMPORTANCE Failure of bone fracture healing occurs in 5% to 10% of all patients. Nonunion risk is associated with the severity of injury and with the surgical treatment technique, yet progression to nonunion is not fully explained by these risk factors.OBJECTIVE To test a hypothesis that fracture characteristics and patient-related risk factors assessable by the clinician at patient presentation can indicate the probability of fracture nonunion.DESIGN, SETTING, AND PARTICIPANTS An inception cohort study in a large payer database of patients with fracture in the United States was conducted using patient-level health claims for medical and drug expenses compiled for approximately 90.1 million patients in calendar year 2011. The final database collated demographic descriptors, treatment procedures as per Current Procedural Terminology codes; comorbidities as per International Classification of Diseases, Ninth Revision codes; and drug prescriptions as per National Drug Code Directory codes. Logistic regression was used to calculate odds ratios (ORs) for variables associated with nonunion. Data analysis was performed from January 1, 2011, to December 31, 2012, EXPOSURES Continuous enrollment in the database was required for 12 months after fracture to allow sufficient time to capture a nonunion diagnosis. RESULTSThe final analysis of 309 330 fractures in 18 bones included 178 952 women (57.9%); mean (SD) age was 44.48 (13.68) years. The nonunion rate was 4.9%. Elevated nonunion risk was associated with severe fracture (eg, open fracture, multiple fractures), high body mass index, smoking, and alcoholism. Women experienced more fractures, but men were more prone to nonunion. The nonunion rate also varied with fracture location: scaphoid, tibia plus fibula, and femur were most likely to be nonunion. The ORs for nonunion fractures were significantly increased for risk factors, including number of fractures (OR, 2.65; 95% CI, 2.34-2.99), use of nonsteroidal anti-inflammatory drugs plus opioids (OR, 1.84; 95% CI, 1.73-1.95), operative treatment (OR, 1.78; 95% CI, 1.69-1.86), open fracture (OR, 1.66; 95% CI, 1.55-1.77), anticoagulant use (OR, 1.58; 95% CI, 1.51-1.66), osteoarthritis with rheumatoid arthritis (OR, 1.58; 95% CI, 1.38-1.82), anticonvulsant use with benzodiazepines (OR, 1.49; 95% CI, 1.36-1.62), opioid use (OR, 1.43; 95% CI, 1.34-1.52), diabetes (OR, 1.40; 95% CI, 1.21-1.61), high-energy injury (OR, 1.38; 95% CI, 1.27-1.49), anticonvulsant use (OR, 1.37; 95% CI, 1.31-1.43), osteoporosis (OR, 1.24; 95% CI, 1.14-1.34), male gender (OR, 1.21; 95% CI, 1.16-1.25), insulin use (OR, 1.21; 95% CI, 1.10-1.31), smoking (OR, 1.20; 95% CI, 1.14-1.26), benzodiazepine use (OR, 1.20; 95% CI, 1.10-1.31), obesity (OR, 1.19; 95% CI, 1.12-1.25), antibiotic use (OR, 1.17; 95% CI, 1.13-1.21), osteoporosis medication use (OR, 1.17; 95% CI, 1.08-1.26), vitamin D deficiency (OR, 1.14; 95% CI, 1.05-1.22), diuretic use (OR, 1.13; 95% CI, 1.07-1.18), and renal insufficiency (OR, 1.11; 95% CI, 1.04-1.17) (multivariate P < .001 for all).C...

In many cells, stimulation of mitogen-activated protein kinases by both receptor tyrosine kinases and receptors that couple to pertussis toxin-sensitive heterotrimeric G proteins proceed via convergent signaling pathways. Both signals are sensitive to inhibitors of tyrosine protein kinases and require Ras activation via phosphotyrosine-dependent recruitment of Ras guanine nucleotide exchange factors. Receptor tyrosine kinase stimulation mediates ligand-induced receptor autophosphorylation, which creates the initial binding sites for SH2 domain-containing docking proteins. However, the mechanism whereby G protein-coupled receptors mediate the phosphotyrosine-dependent assembly of a mitogenic signaling complex is poorly understood. We have studied the role of Src family nonreceptor tyrosine kinases in G protein-coupled receptor-mediated tyrosine phosphorylation in a transiently transfected COS-7 cell system. Stimulation of G i -coupled lysophosphatidic acid and ␣2A adrenergic receptors or overexpression of G␤1␥2 subunits leads to tyrosine phosphorylation of the Shc adapter protein, which then associates with tyrosine phosphoproteins of approximately 130 and 180 kDa, as well as Grb2. The 180-kDa Shc-associated tyrosine phosphoprotein band contains both epidermal growth factor (EGF) receptor and p185 neu . 3-5-fold increases in EGF receptor but not p185 neu tyrosine phosphorylation occur following G i -coupled receptor stimulation. Inhibition of endogenous Src family kinase activity by cellular expression of a dominant negative kinase-inactive mutant of c-Src inhibits G␤1␥2 subunit-mediated and G icoupled receptor-mediated phosphorylation of both EGF receptor and Shc. Expression of Csk, which inactivates Src family kinases by phosphorylating the regulatory carboxyl-terminal tyrosine residue, has the same effect. The G i -coupled receptor-mediated increase in EGF receptor phosphorylation does not reflect increased EGF receptor autophosphorylation, assayed using an autophosphorylation-specific EGF receptor monoclonal antibody. Lysophosphatidic acid stimulates binding of EGF receptor to a GST fusion protein containing the c-Src SH2 domain, and this too is blocked by Csk expression. These data suggest that G␤␥ subunitmediated activation of Src family nonreceptor tyrosine kinases can account for the G i -coupled receptor-mediated tyrosine phosphorylation events that direct recruitment of the Shc and Grb2 adapter proteins to the membrane.The low molecular weight G protein Ras functions as a signaling intermediate in many pathways involved in the regulation of cellular mitogenesis and differentiation. Ras activation by growth factor receptors that possess intrinsic tyrosine kinase activity follows ligand-induced phosphorylation of specific docking sites on the receptor itself or adapter proteins, such as Shc and insulin receptor substrate-1, which serve to recruit Ras guanine nucleotide exchange factors to the plasma membrane (1, 2). Recently, several receptors that couple to heterotrimeric G proteins, including the lysoph...

Diagnostic Level IV. See Instructions to Authors for a complete description of levels of evidence.

Many receptors that couple to heterotrimeric guanine-nucleotide binding proteins (G proteins) have been shown to mediate rapid activation of the mitogen-activated protein kinases Erk1 and Erk2. In different cell types, the signaling pathways employed appear to be a function of the available repertoire of receptors, G proteins, and effectors. In HEK-293 cells, stimulation of either ␣1B-or ␣2A-adrenergic receptors (ARs) leads to rapid 5-10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the ␣2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a G␤␥ subunit complex sequestrant peptide (␤ARK1ct) and dominantnegative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (⌬N-Raf). Erk1/2 phosphorylation in response to ␣1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or ⌬N-Raf, but not by coexpression of ␤ARK1ct or by pretreatment with pertussis toxin. The ␣1B-and ␣2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca 2؉ chelation, and inhibitors of protein-tyrosine kinases. Overexpression of a dominant-negative mutant of c-Src or of the negative regulator of c-Src function, Csk, results in attenuation of the ␣1B-AR-and ␣2A-AR-mediated Erk1/2 signals. Chemical inhibitors of calmodulin, but not of PKC, and overexpression of a dominant-negative mutant of the protein-tyrosine kinase Pyk2 also attenuate mitogenactivated protein kinase phosphorylation after both ␣1B-and ␣2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium-, and tyrosine kinase-dependent pathway for both G i -and G q/11 -coupled receptors. These results indicate that in HEK-293 cells, the G␤␥ subunit-mediated ␣2A-AR-and the G␣ q/11 -mediated ␣1B-AR-coupled Erk1/2 activation pathways converge at the level of phospholipase C. These data suggest that calcium-calmodulin plays a central role in the calcium-dependent regulation of tyrosine phosphorylation by G protein-coupled receptors in some systems.

32 P]GTP into G␣ s (1.57 ؎ 0.12; n ‫؍‬ 3) and G␣ i (1.68 ؎ 0.13; n ‫؍‬ 4) in adipocyte plasma membranes, directly demonstrating that ␤ 3 AR stimulation results in G i -GTP exchange. The ␤ 3 AR-stimulated increase in 8-azido-[ 32 P]GTP labeling of G␣ i was equivalent to that obtained with the A 1 -adenosine receptor agonist N 6 -cyclopentyladenosine (1.56 ؎ 0.07; n ‫؍‬ 4), whereas inclusion of unlabeled GTP (100 M) eliminated all binding. Stimulation of the ␤ 3 AR in 3T3-F442A adipocytes led to a 2-3-fold activation of mitogen-activated protein (MAP) kinase, as measured by extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation. Pretreatment of cells with pertussis toxin (PTX) eliminated MAP kinase activation by ␤ 3 AR, demonstrating that this response required receptor coupling to G i . Expression of the human ␤ 3 AR in HEK-293 cells reconstituted the PTX-sensitive stimulation of MAP kinase, demonstrating that this phenomenon is not exclusive to adipocytes or to the rodent ␤ 3 AR. ERK1/2 activation by the ␤ 3 AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive ␤ 3 AR coupling to G i proteins serves both to restrain G s -mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade.Long before the discovery of the ␤ 3 AR and its recognition as a unique, adipocyte-specific receptor controlling lipolysis and thermogenesis, Rodbell and colleagues (1) made the observation that there was an unusual, biphasic stimulation of cAMP production in adipocytes in response to the ␤-adrenergic receptor agonist isoproterenol. Depending upon the concentration of GTP in the assay, isoproterenol could either stimulate or inhibit adenylyl cyclase activity in adipocyte plasma membranes. Murayama and Ui (2) showed that this inhibitory phase could be relieved by pretreatment of adipocytes with pertussis toxin (PTX).1 This curious observation lay fallow until studied later in greater detail by Bégin-Heick (3-5). However, it was not until the cloning and characterization of the ␤ 3 AR gene and the development of selective ␤ 3 AR agonists (6, 7) that it was postulated that this novel adipocyte-specific ␤AR may be responsible for the biphasic adenylyl cyclase response in adipocytes (8). We have previously noted that despite the relatively high level of expression of the ␤ 3 AR in adipocytes, the efficiency of coupling of the ␤ 3 AR to stimulation of adenylyl cyclase is low (9). However, there has been no clear biochemical demonstration of physical coupling of the ␤ 3 AR to G i , other than comparative functional experiments in the presence or absence of PTX (10), nor has there been any indication of what additional second messenger pathway may be activated as a consequence of this putative coupling of ␤ 3 AR to G i .Recently, many G protein-coupled receptors have been shown to mediate cellular growth or differentiation responses through the activation ...

The Ras-dependent activation of Erk kinases by G protein-coupled receptors (GPCRs) is thought to involve tyrosine phosphorylation of docking proteins that serve as scaffolds for the plasma membrane recruitment of Ras guanine nucleotide exchange factors, such as the Grb2-mSos complex. We have investigated the role of two GPCR-regulated tyrosine phosphoproteins, p125 FAK (FAK) and Shc, in the Ras-dependent activation of Erk kinases by endogenously expressed GPCRs in Rat 1a fibroblasts. Several lines of evidence suggest that tyrosine phosphorylation of FAK and Shc are independently regulated. The GPCRs for lysophosphatidic acid (LPA), thrombin, and bombesin mediate equivalent increases in FAK tyrosine phosphorylation and FAK-Grb2 association. In contrast, only LPA and thrombin receptors significantly stimulate Shc tyrosine phosphorylation and Shc-Grb2 complex formation. Tyrosine phosphorylation of FAK is pertussis toxin-insensitive, can be mimicked by calcium ionophore, and is inhibited by treatment with cytochalasin D, which depolymerizes the actin cytoskeleton. In contrast, tyrosine phosphorylation of Shc is inhibited by pertussis toxin treatment, is not induced by calcium ionophore, and is insensitive to cytochalasin D. In each case, the rapid stimulation of Erk 1/2 correlates with tyrosine phosphorylation of Shc but not of FAK. The dissociation of FAK-Grb2 complex formation from receptor-mediated activation of Erk 1/2 indicates that recruitment of Grb2-mSos to the plasma membrane is not sufficient to mediate rapid Erk activation. Using four mechanistically distinct inhibitors of clathrin-mediated endocytosis, concanavalin A, hypertonic medium, depletion of intracellular potassium, and monodansylcadaverine, we find that GPCR-mediated Erk 1/2 activation is also endocytosis-dependent. Thus, we propose that an additional step involving vesiclemediated endocytosis is required for the rapid, Ras-dependent activation of Erk kinases in fibroblasts.The regulation of gene expression by many types of cell surface receptor involves the Ras-dependent activation of Erk kinases. Receptors as structurally diverse as growth factor receptors that possess intrinsic ligand-stimulated tyrosine kinase activity (RTKs), 1 integrins, which mediate cell surface adhesion, and seven transmembrane-spanning GPCRs have been shown to mediate Ras-dependent growth regulatory signals.The mechanisms whereby such distinct receptors regulate Ras activity are diverse. In each case, however, Ras activation is preceded by the recruitment of Ras guanine nucleotide exchange factors (GEFs) into a plasma membrane-associated signaling complex, where they activate Ras by catalyzing Ras-GTP for Ras-GDP exchange. Ras-GEF recruitment is the consequence of receptor-mediated stimulation of tyrosine protein kinases, which phosphorylate adapter proteins to create tyrosine phosphoprotein scaffolds on the plasma membrane. The individual components of the complex, including Ras-GEFs and other catalytic signaling proteins, are recruited via specific interactions betwe...