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J. Biol. Chem., Vol. 282, Issue 19, 14038-14047, May 11, 2007

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Sequence Dependence and Differential Expression of G5 Subunit Isoforms of the Heterotrimeric G Proteins Variably Processed after Prenylation in Mammalian Cells^*

From the Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425

Between 1 and 2% of proteins coded for in the human genome, including all G protein subunits, are predicted to be prenylated. Subsequently, prenylated proteins are proteolytically cleaved at the C terminus and carboxymethylated. These reactions are generally obligatory events required for functional expression of prenylated proteins. The biological role of prenyl substrates has made these reactions significant targets for anticancer drug development. Understanding the enzymology of this pathway will be key to success for this strategy. When G1, -2, -4, -10, -11, -12, and -13 were expressed in HEK293 cells they were completely processed according to the current understanding of the prenylation reaction. In contrast, G5 was processed to two forms; a minor one, fully processed as predicted, and a major one that was prenylated without further processing. When the Ca₁a₂X motif of G5, CSFL, was exchanged for that of G2, CAIL, G5 was completely processed. Conversely, G2-SFL was incompletely processed. Differential processing of G5 was found due to the presence of an aromatic amino acid in its Ca₁a₂X motif. Retrieving endogenous G subunits from HEK293 or Neuro-2a cells with FLAG-G constructs identified multiple G subunits by mass spectrometry in either cell, but in both cases the most prominent one was G5 expressed without C-terminal processing after prenylation. This work indicates that post-prenylation reactions can generate multiple products determined by the C-terminal Ca₁a₂X motif. Within the human genome 10% of predicted prenylated proteins have aromatic amino acids in their Ca₁a₂X sequence and would likely generate the prenylation pattern described here.

Received for publication, February 15, 2007 , and in revised form, March 12, 2007.

^* This work was supported in part by National Institutes of Health Grant DK37219 and by Institutional support of the Medical University of South Carolina (MUSC) Mass Spectrometry Facility and the MUSC DNA Sequencing Facility. 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.

¹ To whom correspondence should be addressed: Dept. of Pharmacology, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29464. Tel.: 843-792-3209; Fax: 843-792-2475; E-mail: hildebjd{at}musc.edu.

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