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J. Biol. Chem., Vol. 282, Issue 42, 30363-30372, October 19, 2007
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From the Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259
Oligomerization of the Class II G protein-coupled secretin receptor has been reported, but the molecular basis for this and its functional significance have not been determined. In the current work, we have examined the possible contribution of each of the transmembrane (TM) segments of this receptor to its homo-oligomerization, using the method of competitive disruption screening for inhibition of receptor bioluminescence resonance energy transfer signal. TM IV was the only segment that was found to disrupt receptor bioluminescence resonance energy transfer. Evaluation of predicted interhelical and lipid-exposed faces of this TM segment demonstrated that its lipid-exposed face represented the determinant for oligomerization. This was further confirmed by mutagenesis of the intact secretin receptor. Morphological FRET was utilized to demonstrate that secretin receptor oligomerization occurred at the cell surface and that this oligomerization was disrupted by mutating Gly243 and Ile247, key residues within the lipid-exposed face of TM IV. Although disruption of the receptor oligomerization interface had no effect on secretin binding parameters, it reduced the ability of secretin to stimulate intracellular cAMP. This supports a clear functional effect of oligomerization of this receptor. Such an effect might be particularly relevant to clinical situations in which this receptor is overexpressed, such as in certain neoplasms.
Received for publication, March 19, 2007 , and in revised form, August 17, 2007.
* This work was supported by National Institutes of Health Grant DK46577 and funds from the Fiterman Foundation and the Mayo Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S2.
1 To whom correspondence should be addressed: Mayo Clinic, 13400 East Shea Blvd., Scottsdale, AZ 85259. Tel.: 480-301-6650; Fax: 480-301-6969; E-mail: miller{at}mayo.edu.
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