Gregory J. Della Rocca

Essential Role for G Protein-coupled Receptor Endocytosis in the Activation of Mitogen-activated Protein Kinase

The classical paradigm for G protein-coupled receptor (GPCR) signal transduction involves the agonist-dependent interaction of GPCRs with heterotrimeric G proteins at the plasma membrane and the subsequent generation, by membrane-localized effectors, of soluble second messengers or ion currents. Termination of GPCR signals follows G protein-coupled receptor kinase (GRK)- and β-arrestin-mediated receptor uncoupling and internalization. Here we show that these paradigms are inadequate to account for GPCR-mediated, Ras-dependent activation of the mitogen-activated protein (MAP) kinases Erk1 and -2. In HEK293 cells expressing dominant suppressor mutants of β-arrestin or dynamin, β2-adrenergic receptor-mediated activation of MAP kinase is inhibited. The inhibitors of receptor internalization specifically blocked Raf-mediated activation of MEK. Plasma membrane-delimited steps in the GPCR-mediated activation of the MAP kinase pathway, such as tyrosine phosphorylation of Shc and Raf kinase activation by Ras, are unaffected by inhibitors of receptor internalization. Thus, GRKs and β-arrestins, which uncouple GPCRs and target them for internalization, function as essential elements in the GPCR-mediated MAP kinase signaling cascade.

Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors. Convergence of Gi- and Gq-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase.

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 α1B- or α2A-adrenergic receptors (ARs) leads to rapid 5–10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the α2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a Gβγ subunit complex sequestrant peptide (βARK1ct) and dominant-negative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (ΔN-Raf). Erk1/2 phosphorylation in response to α1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or ΔN-Raf, but not by coexpression of βARK1ct or by pretreatment with pertussis toxin. The α1B- and α2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca2+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 α1B-AR- and α2A-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 mitogen-activated protein kinase phosphorylation after both α1B- and α2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium-, and tyrosine kinase-dependent pathway for both Gi- and Gq/11-coupled receptors. These results indicate that in HEK-293 cells, the Gβγ subunit-mediated α2A-AR- and the Gαq/11-mediated α1B-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.

Gβγ Subunits Mediate Src-dependent Phosphorylation of the Epidermal Growth Factor Receptor A SCAFFOLD FOR G PROTEIN-COUPLED RECEPTOR-MEDIATED Ras ACTIVATION

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 Gi-coupled lysophosphatidic acid and α2A adrenergic receptors or overexpression of Gβ1γ2 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 p185neu. 3-5-fold increases in EGF receptor but not p185neu tyrosine phosphorylation occur following Gi-coupled receptor stimulation. Inhibition of endogenous Src family kinase activity by cellular expression of a dominant negative kinase-inactive mutant of c-Src inhibits Gβ1γ2 subunit-mediated and Gi-coupled 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 Gi-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 Gβγ subunit-mediated activation of Src family nonreceptor tyrosine kinases can account for the Gi-coupled receptor-mediated tyrosine phosphorylation events that direct recruitment of the Shc and Grb2 adapter proteins to the membrane.

Clinical presentation of patients with tears of the acetabular labrum

BACKGROUND The clinical presentation of a labral tear of the acetabulum may be variable, and the diagnosis is often delayed. We sought to define the clinical characteristics associated with symptomatic acetabular labral tears by reviewing a group of patients who had an arthroscopically confirmed diagnosis. METHODS We retrospectively reviewed the records for sixty-six consecutive patients (sixty-six hips) who had a documented labral tear that had been confirmed with hip arthroscopy. We had prospectively recorded demographic factors, symptoms, physical examination findings, previous treatments, functional limitations, the manner of onset, the duration of symptoms until the diagnosis of the labral tear, other diagnoses offered by health-care providers, and other surgical procedures that these patients had undergone. Radiographic abnormalities and magnetic resonance arthrography findings were also recorded. RESULTS The study group included forty-seven female patients (71%) and nineteen male patients (29%) with a mean age of thirty-eight years. The initial presentation was insidious in forty patients, was associated with a low-energy acute injury in twenty, and was associated with major trauma in six. Moderate to severe pain was reported by fifty-seven patients (86%), with groin pain predominating (sixty-one patients; 92%). Sixty patients (91%) had activity-related pain (p < 0.0001), and forty-seven patients (71%) had night pain (p = 0.0006). On examination, twenty-six patients (39%) had a limp, twenty-five (38%) had a positive Trendelenburg sign, and sixty-three (95%) had a positive impingement sign. The mean time from the onset of symptoms to the diagnosis of a labral tear was twenty-one months. A mean of 3.3 health-care providers had been seen by the patients prior to the definitive diagnosis. Surgery on another anatomic site had been recommended for eleven patients (17%), and four had undergone an unsuccessful operative procedure prior to the diagnosis of the labral tear. At an average of 16.4 months after hip arthroscopy, fifty-nine patients (89%) reported clinical improvement in comparison with the preoperative status. CONCLUSIONS The clinical presentation of a patient who has a labral tear may vary, and the correct diagnosis may not be considered initially. In young, active patients with a predominant complaint of groin pain with or without a history of trauma, the diagnosis of a labral tear should be suspected and investigated as radiographs and the history may be nonspecific for this diagnosis. LEVEL OF EVIDENCE Diagnostic Level IV. See Instructions to Authors for a complete description of levels of evidence.

Pleiotropic Coupling of G Protein-coupled Receptors to the Mitogen-activated Protein Kinase Cascade ROLE OF FOCAL ADHESIONS AND RECEPTOR TYROSINE KINASES

G protein-coupled receptors (GPCRs) initiate Ras-dependent activation of the Erk 1/2 mitogen-activated protein kinase cascade by stimulating recruitment of Ras guanine nucleotide exchange factors to the plasma membrane. Both integrin-based focal adhesion complexes and receptor tyrosine kinases have been proposed as scaffolds upon which the GPCR-induced Ras activation complex may assemble. Using specific inhibitors of focal adhesion complex assembly and receptor tyrosine kinase activation, we have determined the relative contribution of each to activation of the Erk 1/2 cascade following stimulation of endogenous GPCRs in three different cell types. The tetrapeptide RGDS, which inhibits integrin dimerization, and cytochalasin D, which depolymerizes the actin cytoskeleton, disrupt the assembly of focal adhesions. In PC12 rat pheochromocytoma cells, both agents block lysophosphatidic acid (LPA)- and bradykinin-stimulated Erk 1/2 phosphorylation, suggesting that intact focal adhesion complexes are required for GPCR-induced mitogen-activated protein kinase activation in these cells. In Rat 1 fibroblasts, Erk 1/2 activation via LPA and thrombin receptors is completely insensitive to both agents. Conversely, the epidermal growth factor receptor-specific tyrphostin AG1478 inhibits GPCR-mediated Erk 1/2 activation in Rat 1 cells but has no effect in PC12 cells. In HEK-293 human embryonic kidney cells, LPA and thrombin receptor-mediated Erk 1/2 activation is partially sensitive to both the RGDS peptide and tyrphostin AG1478, suggesting that both focal adhesion and receptor tyrosine kinase scaffolds are employed in these cells. The dependence of GPCR-mediated Erk 1/2 activation on intact focal adhesions correlates with expression of the calcium-regulated focal adhesion kinase, Pyk2. In all three cell types, GPCR-stimulated Erk 1/2 activation is significantly inhibited by the Src kinase inhibitors, herbimycin A and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1), suggesting that Src family nonreceptor tyrosine kinases represent a point of convergence for signals originating from either scaffold.

G Protein-coupled Receptors Mediate Two Functionally Distinct Pathways of Tyrosine Phosphorylation in Rat 1a Fibroblasts Shc PHOSPHORYLATION AND RECEPTOR ENDOCYTOSIS CORRELATE WITH ACTIVATION OF Erk KINASES

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, p125FAK (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 vesicle-mediated endocytosis is required for the rapid, Ras-dependent activation of Erk kinases in fibroblasts.

The β3-Adrenergic Receptor Activates Mitogen-activated Protein Kinase in Adipocytes through a Gi-dependent Mechanism

Promiscuous coupling between G protein-coupled receptors and multiple species of heterotrimeric G proteins provides a potential mechanism for expanding the diversity of G protein-coupled receptor signaling. We have examined the mechanism and functional consequences of dual Gs/Gi protein coupling of the β3-adrenergic receptor (β3AR) in 3T3-F442A adipocytes. The β3AR selective agonist disodium (R,R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in cAMP production in adipocyte plasma membrane preparations, and pretreatment of cells with pertussis toxin resulted in a further 2-fold increase in cAMP production by CL316,243. CL316,243 (5 μm) stimulated the incorporation of 8-azido-[32P]GTP into Gαs (1.57 ± 0.12; n = 3) and Gαi (1.68 ± 0.13;n = 4) in adipocyte plasma membranes, directly demonstrating that β3AR stimulation results in Gi-GTP exchange. The β3AR-stimulated increase in 8-azido-[32P]GTP labeling of Gαi was equivalent to that obtained with the A1-adenosine receptor agonist N 6-cyclopentyladenosine (1.56 ± 0.07; n = 4), whereas inclusion of unlabeled GTP (100 μm) eliminated all binding. Stimulation of the β3AR 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 β3AR, demonstrating that this response required receptor coupling to Gi. Expression of the human β3AR 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 β3AR. ERK1/2 activation by the β3AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive β3AR coupling to Gi proteins serves both to restrain Gs-mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade.

Serotonin 5-HT1A receptor-mediated Erk activation requires calcium/calmodulin-dependent receptor endocytosis.

Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin (5-hydroxytryptamine (5-HT)) 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation was attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation, because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase had no effect upon 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis was required for 5-HT1A receptor-mediated Erk1/2 activation, we postulated a role for CAM in receptor endocytosis. Inhibition of receptor endocytosis by use of sequestration-defective mutants of β-arrestin1 and dynamin attenuated 5-HT1Areceptor-stimulated Erk1/2 activation. Inhibition of CAM prevented agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. We conclude that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects its role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2.

ACTIVATION OF EXTRACELLULAR SIGNAL-REGULATED KINASE IN HUMAN PROSTATE CANCER

PURPOSE To investigate the level of expression, activation state, and functional significance of extracellular signal regulated kinase (ERK) in prostate cancer. MATERIALS AND METHODS Human prostate tissue samples (n = 22) were obtained from patients undergoing radical prostatectomy for localized adenocarcinoma of the prostate (n = 16, age range 44 to 72 years) or normal prostate specimens (n = 6, age ranges 19 to 47 years) obtained from rapid autopsy. Immunoblots, in vitro kinase assays, and immunohistochemistry were used to determine the expression and activation state of ERK in human prostate cancer. RESULTS Immunoblot and in vitro kinase assays demonstrated a 15-fold increase in ERK activation in prostate cancer specimens compared with normal human prostate tissue; however, ERK expression levels were only 1.3-fold higher in cancer. Immunohistochemical analysis demonstrated similar expression of ERK in cancer and normal tissues; however, phosphorylated ERK demonstrated greater intensity in the cancer specimens. Experiments conducted on a prostate cancer cell line demonstrated that EGF induced activation of ERK and cellular proliferation was partially inhibited by PD98059, a chemical inhibitor of the immediate upstream signaling component responsible of activation of ERK. CONCLUSIONS Collectively, these data demonstrate a dramatic increase in ERK activation in prostate cancer compared with normal prostate tissue and suggest that inhibitors designed to target this signal transduction cascade might have therapeutic benefit in the treatment of prostate cancer.

Acetabular and femoral radiographic abnormalities associated with labral tears

The purpose of our study was to define the incidence of acetabular and femoral osseous abnormalities associated with symptomatic acetabular labral tears. We reviewed the radiographs of 78 patients treated arthroscopically for labral tears and 22 patients with asymptomatic hips for comparison. Overall, 49% of patients with labral tears had at least one radiographic abnormality (17% acetabular, 14% femoral, and 18% both). Hip dysplasia was more prevalent in patients with labral tears (36%) compared with control subjects (0%). A decreased head-neck offset was present in 18% of patients with labral tears versus 5% of the control subjects. An anterolateral prominence at the femoral head-neck junction, creating an aspherical femoral head, was present in 29% of patients with labral tears. Sixty-one percent of those patients also met criteria for dysplasia and/or decreased head-neck offset. A retroverted acetabulum was present in 12% of patients with labral tears and none of the control subjects. Osteoarthritis was more common in patients with labral tears (33%) than in control subjects (9%). Because acetabular and femoral osseous abnormalities commonly are associated with labral tears, recognition of these abnormalities is important to optimize surgical treatment of patients with symptomatic labral disease. Level of Evidence: Diagnostic study, Level II (development of diagnostic criteria on consecutive patients-with universally applied reference “gold” standard). See the Guidelines for Authors for a complete description of levels of evidence.