The ErbB2/ErbB3 heregulin co-receptor has been shown to couple to phosphoinositide (PI) 3-kinase in a heregulin-dependent manner. The recruitment and activation of PI 3-kinase by this co-receptor is presumed to occur via its interaction with phosphorylated TyrXaa-Xaa-Met (YXXM) motifs occurring in the ErbB3 C terminus. In this study, mutant ErbB3 receptor proteins expressed in COS7 cells were used to investigate PI 3-kinase-dependent signaling pathways activated by the ErbB2/ErbB3 co-receptor. We observed that a mutant ErbB3 protein with each of its six YXXM motifs containing a Tyr 3 Phe substitution was unable to bind either the p85 regulatory or p110 catalytic subunit of PI 3-kinase. However, restoration of a single YXXM motif was sufficient to mediate association with the PI 3-kinase holoenzyme, although at a lower level than wild-type ErbB3. When ErbB3 YXXM motifs were restored in pairs, evidence for cooperativity between two, those incorporating Tyr-1273 and Tyr-1286, was observed. Interestingly, we have shown that an apparent association of PI 3-kinase activity with ErbB2/Neu was due to the residual presence of ErbB3 in ErbB2 immunoprecipitates. The necessity of ErbB3 association with PI 3-kinase for downstream signaling to the effector kinase Akt was also investigated. Here, the heregulin-dependent translocation of Akt to the plasma membrane and its subsequent activation was observed in intact NIH-3T3 fibroblasts. Recruitment of PI 3-kinase to ErbB3 was required for both activities, and it appeared that ErbB2 activation alone was not sufficient to activate PI 3-kinase signaling in these cells.The type I subfamily of receptor protein-tyrosine kinases is composed of four members: the prototypical epidermal growth factor receptor (ErbB1/HER1), ErbB2 (HER2/Neu), ErbB3 (HER3), and ErbB4 (HER4). Interestingly, this family of receptors can generate a wide variety of cellular signals by mixing and matching to form various co-receptor signaling complexes (1-4). Of the various heterodimers and homodimers formed, the ErbB2/ErbB3 dimer constitutes a high affinity co-receptor for heregulin (5), which is capable of potent mitogenic signaling.In particular, ErbB3 has been characterized as a major mediator of heregulin-dependent activation of the phosphoinositide (PI) 1 3-kinase pathway (6 -14). One general mechanism of recruitment and activation of PI 3-kinase involves the binding of the tandem Src homology 2 (SH2) domains of its p85 regulatory subunit to phosphorylated YXXM motifs found in signaling proteins (15, 16). ErbB3 is particularly well adapted to mediate PI 3-kinase signaling, because it contains in its Cterminal phosphorylation domain six such consensus p85 binding motifs (17). Two previous studies have investigated the role of these YXXM motifs in ErbB3 signaling. One study showed that phosphopeptides containing the various ErbB3 YXXM motifs could inhibit p85 association with ErbB3 (9). Additionally, we have shown via the yeast two-hybrid system that these phosphorylated motifs can directly associate with th...

ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr-->Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3.

The role of protein tyrosine kinase activity in ErbB3-mediated signal transduction was investigated. ErbB3 was phosphorylated in vivo in response to either heregulin (HRG) in cells expressing both ErbB3 and ErbB2, or epidermal growth factor (EGF) in cells expressing both ErbB3 and EGF receptor. A recombinant receptor protein (ErbB3-K/M, in which K/M stands for Lys-->Met amino acid substitution) containing an inactivating mutation in the putative ATP-binding site was also phosphorylated in response to HRG and EGF. Both the wild-type ErbB3 and mutant ErbB3-K/M proteins transduced signals to phosphatidylinositol 3-kinase, Shc and mitogen-activated protein kinases. Separate kinase-inactivating mutations in the EGF receptor and ErbB2 proteins abolished ErbB3 phosphorylation and signal transduction activated by EGF and HRG respectively. Hence the protein tyrosine kinase activity necessary for growth factor signalling via the ErbB3 protein seems to be provided by coexpressed EGF and ErbB2 receptor proteins.

STUDY DESIGN Clinical measurement, cross-sectional. OBJECTIVES To establish a set of normal values for diaphragm thickening with tidal breathing in healthy subjects. BACKGROUND Normal values for diaphragm contractility, as imaged sonographically, have not been described, despite the known role of the diaphragm in contributing to spinal stability. If the normal range of diaphragm contractility can be defined in a reliable manner, ultrasound has the potential to be used clinically and in research as a biofeedback tool to enhance diaphragm activation/contractility. METHODS B-mode ultrasound was performed on 150 healthy subjects to visualize and measure hemi-diaphragm thickness on each side at resting inspiration and expiration. Primary outcome measures were hemi-diaphragm thickness and thickening ratio, stratified for age, gender, and body mass index. Interrater and intrarater reliability were also measured. RESULTS Normal thickness of the diaphragm at rest ranged from 0.12 to 1.18 cm, with slightly greater thickness in men but no effect of age. Average ± SD change in thickness from resting expiration to resting inspiration was 20.0% ± 15.5% on the right and 23.5% ± 24.4% on the left; however, almost one third of healthy subjects had no to minimal diaphragm thickening with tidal breathing. CONCLUSION There is wide variability in the degree of diaphragm contractility during quiet breathing. B-mode ultrasound appears to be a reliable means of determining the contractility of the diaphragm, an important muscle in spinal stability. Further studies are needed to validate this imaging modality as a clinical tool in the neuromuscular re-education of the diaphragm to improve spinal stability in both healthy subjects and in patients with low back pain.

Interprofessional education (IPE) in clinical practice is believed to improve outcomes in health care delivery. Integrating teaching and learning objectives through cross discipline student interaction in basic sciences has the potential to initiate interprofessional collaboration at the early stages of health care education. Student attitudes and effectiveness of IPE in the context of a combined gross anatomy course for first-year students in Doctor of Physical Therapy (DPT) and Doctor of Medicine (MD) degrees curricula were evaluated. Integrated teams of MD and DPT students participated in part of the gross anatomy dissection course at Mayo Medical School. A survey was administered to 42 MD and 28 DPT students that assessed their attitudes toward IPE and cooperation among health care professionals. Pre- and post-experience surveys were evaluated. Positive comments were related to opportunities for developing a better understanding of the nature and scope of each other's programs, encouraging teamwork and communication, mutual respect, and reducing the perceptual divide between disciplines. Ninety-two percent of the students agreed that interprofessional learning would help them in becoming a more effective member of the health care team. This initial experience with IPE in gross anatomy provides a basis for continued development of interdisciplinary educational strategies.