604 A Comparative Study of Patient Perceptions and Screening Preferences for Stool- Based DNA Testing (SBDNA), Fecal Occult Blood Testing (FOBT) or Colonoseopy (CS) Paul C. Schroy III, Timothy C. Heeren Background: Poor patient acceptance is a major barrier to effective colorectal cancer (CRC) greening. SBDNA is a novel CRC screening strategy that offers a convenient, non-invasive and potentially more acceptable alternative to existing screening tests. The objective of this studywas to indirectly assess patient acceptance by comparing patient perceptions of SBDNA, FOBT and CS and eliciting preferences. Methods: A prospective survey was conducted amongpreviously unscreened, asymptomatic average-risk subjects 50 years of age and older panicipating in a multi-center comparison of SBDNA (PreGen-Phis TM) and FOBT (Hemoccult If) for detecting CRC neoplasia. Evahiable subjects completed a 25-item questionnaire within 48hrs after undergoing a screening colonoscopy, which served as the reference standard for SBDNA and FOBT test performance. Respondents were asked to rate each of the 3 screening tests on a variety of features using a 5-point ordinal scale and select a preferred strategy. Results:A total of 2,388 subjects completed the survey. SBDNA received higher mean ratings (p<0.001) than FOBT for simplicity of the stool sample collection process, comfort, perceived accuracyand likelihood of repeating the test if recommended by their doctor. SBDNA and FOBT received similar ratings for simplicity of prep instructions, lost time from work or otheractivities, perceived invasiveness, perceived embarrassment and perceived anxiety over the prep and test itself. FOBT was never rated higher than SBDNA with respect to any feature. When compared with CS, SBDNA received higher ratings (p<~0.001) for all of the alorementioned test features except accuracy, where CS was rated higher (p<~O.001). Overall, a higher percentage (p<0.001) of patients preferred SBDNA (50.1%) to both FOBT (34.6%) and CS (15.3%) for their routine screening program. Conclusions: Average risk patients perceive SBDNA to have a number of advantages over both FOBT and CS. Moreover, our data suggest that SBDNA may be a preferred strategy for routine CRC screening. 605 The Extracellular Domain of Receptor Activity-Modifying Protein-1 (RAMP-l) is Sufficient for Calcitonin Receptor-Like Receptor (CRLR) Function Timothy]. Fitzsimmons, Xilin Zhao, Stephen A. Wank Background:Calcitonin Gene-Related Peptide (CGRP) is a neuropeptide found in the central andperipheral nervous systems. CGRE mediates sensory neurotransmission, decreases vascu- lar tone, gastrointestinal motility and secretion, and inhibits the action of insulin on carbohy- drate metabolism. A functional CGRP receptor requires dimerization of Calcitonin Receptor- hke Receptor (CRLR) with Receptor Activity-Modifying Protein-1 (RAMP-I). Purpose: To determine the function of the three domains (extracelhilar (ECD), transmembrane (TM), and tail domains) of human RAMP-1. Methods: Three mutants were constructed: 1) RAMP- 1 without the cytoplasmic tail, 2) a chimera consisting of the ECD of RAMP-1 and the TM and tail of the Pfatelet Derived Growth Factor Receptor (PDGFR) and 3) the ECD of RAMP- 1 alone. These RAMP-1 mutants were examined for their ability to associate with CRLR to effectCGRP stimulated cAMP accumulation and esI-CGRP binding, as well as CRLR traffick- ing and cell surface expression as determined by flow cytometry and immunoblotting. Results: All RAMP-1 mutants were able to associate with CRLR with full efficacy for CGRP stimulated cAMP accumulation. However, the RAMP-1/PDGFR chimera demonstrated a 10- f01d decrease in potency for CGRP signaling and a similar decrease for CGRP binding. The RAMP-1 ECD mutant had a 4000-fold decrease in potency and binding was too weak to measure. CRLR cell surface expression was retained with all RAMP-1 mutants, although the RAMP-1 mutant consisting of the ECD alone functioned less efficiently and was secreted into the medium. Conclusions: The ECD of RAMP- 1 is sufficient for normal CRLR association and efficacy. The presence of a TM domain and the specific sequence of the RAMP-1 TM domaincontribute to CGRP affinity and potency. The C-terminal tail of RAMP- 1 is unneces- saryfor CRLR function. 606 Neurotensin Release In Response To Protein Kinase C Activation By Phorbol Esters Is Mediated Through Myristoylated Alanine-Rich C Kinase Substrate Phosphorylation Jiag Li, Kathleen L. O'Connor, Leoncio Vergara, Mark R. Hellmich, George H. Greeley Jr, Courtney M Townsend Jr., B. Mark Evers Myristoylated alanine-rich C-hnase substrate (MARCKS) is a cellular substrate for protein kinase C (PKC). Recently, we have shown that PKCa and B play a role in pborbol 12- myristate 13-acetate (PMA)-mediated secretion of neurotensin (NT), a gut peptide which affects GI secretion, motility and growth. In our present study, we determined whether PKC-mediated NT secretion is due to the activation of MARCKS using the novel endocrine cell line BON, which produces and secretes NT peptide. METHODS. To determine the role 0f MARCKSin PKC-mediated NT secretion, stable transfection of bovine MARCKS, (wild- type, myristoylation and phosphorylation site domain (PSD) mutants), was performed in BON cells; NT secretion was measured by RIA. Transloeation of GFP-tagged MARCKS or endogenous MARCKS was observed by time series confocal analysis or subcellular fraction- ation, respectively. Phosphorylation was assessed by Westem blots. RESULTS. Overexpres- sion of wild-type MARCKS resulted in a significant increase in PMA-mediated NT secretion compared with the empty (ie, control) vector. GFP-tagged wild-type and PSD mutant MARCKS proteins were expressed in the membrane but the myristoylation site mutant was detected only in the cytosol. In addition, translocation was detected from membrane to cytosolby time series confocal analysis lmin after PMA addition only in wild-type MARCKS in living cells, demonstratmg the function of MARCKS is myristoylation and PSD phosphory- lation dependent. Pretreatment with PKC inhibitors (Go6983, GFX and Ro31-8220) effec- tivelyinhibited the phosphorylation of MARCKS, suggesting the regulation of PKC on the activity of MARCKS. CONCLUSIONS. We demonstrate, for the first time, that MARCKS protein is involved in PMA-mediated NT secretion in BON ceils. Furthermore, activity of MARCKS is regulated by PKC. These findings provide novel information regarding signaling mechanisms contributing to stimulated gut peptide secretion. 607 Signal Transduction Pathways Regulating Parietal Cell Dedifferentiation Vinzenz Stepan, Saravanan Ramamoorthy, Hildegard Nitscbe, Kilsti Brown, Andrea Todisco The Sonic Hedgehog (Shh) signal transduction pathway regulates cellular growth and differ- entiation. Shh binds to the receptor protein patched (Ptc), relieving the tonic inhibitory effect of Ptr on the transmembrane protein Smoothened (Smo). This allows the activation of downstream targets through the Gli family of transcription factors. We previously reported that prolonged incubation (> 72 h) of purified (>95%) canine gastric parietal cells in primary culture with EGF induces parietal cell dedifferentiation, a process characterized by morphological transformation, inhibition of H+/K+-ATP-ase gene expression and induction of cell proliferation. In this study we explored the role of Shh/Ptc/Smo in the dedifferentiating actions of EGF in the parietal ceils. EGF (10 nM) suppressed the expression of Shh and Ptc but~hot of Smo in the parietal cells after 72~h 0fi~bation. Shh, Ptc and Smo expression were measured by western blots and by immuno~bchemieal staining of the parietal ceils with anti-Shh, -Ptc and -Smo antibodies. We examined the consequences of loss of expression of the inhibitory protein Ptc. Cyclopamine (1jzM) an inhibitor of Smo signaling, reversed EGF-induced parietal cell morphological transformation and it inhibited EGF induction of both cyclin DI and TGF-c~ expression in the parietal cells, measured by westem blots with anti-cyclin D 1 and -TGFa antibodies. Cyclopamine also inhibited MAPK activation, measured by western blots with anti-phospho-MAPK antibodies and it blocked EGF-stimulated parietal cell proliferation, assessed by incorporation of tritiated thymidine into the DNA. To further explore if deregulated Smo signaling affects TGFa expression, we co-transfected the parietal cells with a hiciferase reporter plasmid containing 2813 bases of the human TGFct gene promoter together with a dominant negative Gh2 gene. EGF induced TGFa luciferase activity two-fold, and dominant negative Gli2 inhibited this effect. Since Akt promotes parietal cell differentiation, we examined the effect of Akt on Shh and Ptc expression in EGF-treated, dedifferentiated parietal cells. Adenovirus mediated gene transfer of constitutively active Akt into the dedifferentiated parietal cells, restored the expression of both Shh and Ptc. Accord- ingly, prolonged exposure of the parietal cells to EGF leads to parietal cell dedifferentiation through loss of Ptc and continuous, deregulated signaling from Smo. Akt appears to be a crucial switch for the activation of programs of parietal cell differentiation. 6O8 Cooperation of GQ, GI and G12/13 in Proteig/rl~tnase D Activation and Phosphorylation Induced by Lysophosphattdie Acid Jingahen Ynan, Lee W. Slice, Jennifer Gu, Enrique l~zengurt STUDY PURPOSE: Lysophosphatidic acid (LPA) promotes a broad range of biological responses and multiple molecular events in target cells. Consistent with the stimulation of multiple signaling pathways, LPA has been shown to activate severalheterotrimeric G proteins including Gq, Gi and G13 in Swiss 3T3 cells. Protein kinase D (PKD/PKCIz) is a seilne/ threonine protein kinase with distinct structural, enzymological and regulatory properties. Recently, activation of a number of receptors that couple to heterotilmeric G proteins including those for LPA has been shown to stimulate PKD activation in a variety of cell types. PKC-dependent PKD activation has been identified as an early event in the action of LPA in intact Swiss 3T3, Rat-1 and IEC-6 cells. In order to examine the contribution of different G-protein pathways to LPA-induced PKD activation, we tested the effect of LPA on PKD activity in mutine embryonic cell lines deficient in Gaq/11 (Guq/11 KO cells) or G~ 12/13 (Get12/13 KO cells), and used cells lacking rhodopsin kinase (RIgcells) as a control. RESULTS: In RK and Gut12/13 KO cells, LPA induced PKD activation through a PLC/PKC pathway in a concentration-dependent fashion with mammal stimulation (6-fold for RK cells and 4-fold for Ga12/13 KO cells in autophosphorylation activity) achieved at 3 ~M. In contrast, LPA did not induce any significant increase in PKD activity in Gaq/11 KO cells. However, LPA induced a significantly increased PKD activity when Gaq/11 KO cells were transfected with Gctq. LPA-induced PKD activation was modestly attenuated by prior expo- sure of RK ceils to pertussis toxin (Fix) but almost abolished by the combination treatments of Fix and Clostridium difficile toxin B, a potent inhibitor of Rho. Surprisingly, Fix alone strikingly inhibited LPA-induced PKD activation in a concentration-dependent fashion in Ga12/13 KO cells. Similar results were obtained when activation loop phosphoryfation at Ser-744 was determined using a specific antibody that detects the phosphorylated state of this residue. CONCLUSION: Our results indicate that Gq is necessary but not sufficient to mediate LPA-indnced PKD activation. In addition to Gq, LPA requires additional G-protein pathways to elicit a maximal response with Gi playing a critical role in Ga12/13 KO cells. We conclude that LPA induces PKD activation through Gq, Gi and G12, and propose that PKD activation is a point of convergence in the action of multiple G-protein pathways. 609 Multiple Signaling Pathways for EDG (endothelial differentiation gene) Receptors in Gastric Smooth Muscle Huipmg Zhou, Karnam S. Murthy, Sankar Das, John R. Gilder, Gabriel M. Makhlouf Eight EDG receptors have been cloned, five of which (EDG 1, -3, -5, -6, and 8) are selectively activated by sphingosine-l-phosphate (SIP). We have recently shown that EDG5 receptors are expressed in gastric smooth muscle cells. In other cells types, EDG receptors couple variously to Fix-sensitive and -insensitive G proteins. Aim: To characterize the signahng pathways initiated by S1P in rabbit gastric muscle cells. Methods: G protein activation was determined in freshly dispersed muscle cells. PI hydrolysis and cAMP formation were measured in freshly dispersed and cultured muscle cells and in cultured muscles cells transfected with various Ga minigenes. Muscle contraction was measured by scanning micrometry. Results: SIP stimulated the activities of Gq, G~3, and all three isoforms of G,, A-77 AGA Abstracts