Publications

Current data from in vitro and in vivo animal models indicate that fibronectin-binding integrin receptors expressed by colon cancer cells may regulate tumour growth. While individual members of the beta 1 subfamily of integrins have now been clearly identified in colorectal cancer, little information exists with respect to the alpha V subfamily. In the present study we show that alpha V can associate with multiple and different beta subunits capable of binding fibronectin in this tumour type. This is likely to have functional implications for growth and spread of colorectal cancer.

The olefinic barbiturate secobarbital (SB) is a sedative hypnotic known to be a relatively selective mechanism-based inactivator of rat liver cytochrome P450 2B1. Previous studies have demonstrated that such inactivation results in prosthetic heme destruction and irreversible drug-induced protein modification, events most likely triggered by P450 2B1-dependent oxidative activation of the olefinic pi-bond. However, the precise structure of the SB-modified heme and/or the protein site targeted for attack remained to be elucidated. We have now isolated the SB-heme adduct from P450 2B1 inactivated by [14C]SB in a functionally reconstituted system and structurally characterized it by electronic absorption spectroscopy and tandem collision-induced dissociation (CID), matrix-assisted laser desorption ionization on time of flight (MALDI-TOF), and liquid secondary ion mass spectrometry in the positive mode (+ LSIMS) as the N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX adduct. The [14C]SB-modified 2B1 protein has also been isolated from similar inactivation systems and subjected to lysyl endopeptidase C (Lys-C) digestion and HPLC-peptide mapping. A [14C]SB-modified 2B1 peptide was thus isolated, purified, electrotransferred onto a poly-(vinylidene) membrane, and identified by micro Edman degradation of its first N-terminal 17 residues (S277NH(H)TEFH(H)ENLMISLL293) as the Lys-C peptide domain comprised of amino acids 277-323. This peptide thus includes the peptide domain corresponding to the distal helix I of P450 101, a region highly conserved through evolution, and which is known not only to flank the heme moiety but also to intimately contact the substrates. This finding thus suggests that SB-induced protein modification of P450 2B1 also occurs at the active site and, together with heme N-alkylation, contributes to the SB-induced mechanism-based inactivation of P450 2B1.

We have recently shown that a four-amino acid epitope (VTIL) on the m2 muscarinic receptor (corresponding to Val385, Thr386, Ile389, and Leu390) is essential for Gi/o coupling specificity and Gi/o activation (Liu, J., Conklin, B. R., Blin, N., Yun, J., and Wess, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11642-11646). Because this sequence element is thought to be located at the junction between the third intracellular loop and the sixth transmembrane helix (TM VI), we speculated that agonist binding to the m2 receptor protein results in conformational changes that enable the VTIL motif to interact with Gi/o proteins. To test the hypothesis that such structural changes might involve a relative movement of TM VI toward the cytoplasm, we created a series of mutant m2 muscarinic receptors in which one to four extra Ala residues were inserted into TM VI immediately after Leu390. Based on the geometry of an alpha-helix, such mutations are predicted to "push" the VTIL sequence away from the lipid bilayer. Consistent with our working hypothesis, second messenger assays with transfected COS-7 cells showed that all mutant m2 receptors containing extra Ala residues C-terminal of Leu390 could activate the proper G proteins even in the absence of agonist. However, replacement of the VTIL motif in such constitutively active m2 receptors with the corresponding m3 muscarinic receptor sequence (AALS) or deletion of Ala391 from the wild type m2 receptor completely abolished G protein coupling. Interestingly, introduction of extra Ala residues C-terminal of the AALS motif in the m3 muscarinic receptor completely abolished functional activity. Mutant m2 and m3 receptors that contained extra Ala residues immediately N-terminal of the VTIL and AALS motif, respectively, displayed wild type-like coupling properties. Our data are consistent with a model in which agonist binding to the m2 muscarinic receptor leads to a relative movement of TM VI toward the cytoplasm, thus enabling the adjacent VTIL sequence to interact with the C terminus of Galpha(i/o) subunits.