Major functions of macrophages include maintaining tissue homeostasis and responding to micro-organisms

orgia, United States of America Received February 15, 2011; Accepted March 18, 2011; Published April 12, 2011 Copyright: 2011 Willis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by National Institutes of Health Grant GM 032875 and National Institutes of Health Grant F32 DK10005-03 to C.M.N. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: [email protected] Current address: Array BioPharma, Boulder, Colorado, United States of America Current address: Vanderbilt Program in Drug Discovery, Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States of America Introduction The cyclic AMP-dependent protein kinase holoenzyme exists as an inactive heterotetrameric complex of two catalytic subunits that are bound and inhibited by two dimerized regulatory subunits. Cooperative binding of two cAMP molecules to each R Brivanib web subunit causes the release of active C subunit and leads to downstream cellular changes in the activity of transcription factors, enzymes, ion channels, and many other cellular substrates. The mouse genome encodes four R subunit genes and two C subunit genes . Furthermore, both Ca and Cb genes have alternative splice variants, thus adding to the diversity of PKA signaling pathways. Most tissues constitutively express the a subunits whereas the expression patterns of the b subunits are more restricted. A major obstacle in studies to delineate the role of PKA in specific physiological pathways has been our inability to obtain cell-type specific inhibition or activation of the kinase in vivo. One approach to study the physiological role of PKA in vivo has relied on molecular genetic techniques to disrupt specific PKA subunit genes or overexpress mutant forms of PKA subunits. Each PKA subunit gene has been individually disrupted in mice and despite the widespread expression patterns of R and C isoforms, only the disruption of RIa results in embryonic lethality. Furthermore, in RIb-, RIIa-, and RIIb-null mice, the RIa subunit has the ability to compensate for the loss of other R subunits in several tissues and it has been suggested that RIa serves as a physiological ��buffer��to limit the activity of free C subunit when it exists in excess of R subunit. The R subunits have two cAMP binding sites in the carboxyl terminal domain of the protein and mutations that disrupt cAMP binding to either site inhibit the ability of cAMP to activate the mutant holoenzyme. In the present study, we used gene targeting to introduce a point mutation into exon 11 of the endogenous RIa allele. This point mutation results in a Gly to Asp substitution at amino acid 324 within the site B cAMPbinding domain in RIa and is identical to the site B mutation first characterized in S49 cells. This mutation prevents cAMP from binding to site B and produces a dominant negative phenotype in cell culture. The introduction of a loxP-flanked neomycin resistance cassette and polyadenylation signal into the intron upstream of the site B mutation prevents expression of the mutant RIa allele; however, in the presence of Cre recombinase, excision oforgia, United States of America Received February 15, 2011; Accepted March 18, 2011; Published April 12, 2011 Copyright: 2011 Willis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by National Institutes of Health Grant GM 032875 and National Institutes of Health Grant F32 DK10005-03 to C.M.N. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: [email protected] Current address: Array BioPharma, Boulder, Colorado, United States of America Current address: Vanderbilt Program in Drug Discovery, Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States of America Introduction The cyclic AMP-dependent protein kinase holoenzyme exists as an inactive heterotetrameric complex of two catalytic subunits that are bound and inhibited by two dimerized regulatory subunits. Cooperative binding of two cAMP molecules to each R subunit causes the release of active C subunit and leads to downstream cellular changes in the activity of transcription factors, enzymes, ion channels, and many other cellular substrates. The mouse genome encodes four R subunit genes and two C subunit genes . Furthermore, both Ca and Cb genes have alternative splice variants, thus adding to the diversity of PKA signaling pathways. Most tissues constitutively express the a subunits whereas the expression patterns of the b subunits are more restricted. A major obstacle in studies to delineate the role of PKA in specific physiological pathways has been our inability to obtain cell-type specific inhibition or activation of the kinase in vivo. One approach to study the physiological role of PKA in vivo has relied on molecular genetic techniques to disrupt specific PKA subunit genes or overexpress mutant forms of PKA subunits. Each PKA subunit gene has been individually disrupted in mice and despite the widespread expression patterns of R and C isoforms, only the disruption of RIa results in embryonic lethality. Furthermore, in RIb-, RIIa-, and RIIb-null mice, 17942897 the RIa subunit has the ability to compensate for the loss of other R subunits in several tissues and it has been suggested that RIa serves as a physiological ��buffer��to limit the activity of free C subunit when it exists in excess of R subunit. The R subunits have two cAMP binding sites in the carboxyl terminal domain of the protein and mutations that disrupt cAMP binding to either site inhibit the ability of cAMP to activate the mutant holoenzyme. In the present study, we used gene targeting to introduce a point mutation into exon 11 of the endogenous RIa allele. This point mutation results in a Gly to Asp substitution at amino acid 324 within the site B cAMPbinding domain in RIa and is identical to the site B mutation first characterized in S49 cells. This mutation prevents cAMP from binding to site B and produces a dominant negative phenotype in cell culture. The introduction of a loxP-flanked neomycin resistance cassette and polyadenylation signal into the intron upstream of the site B mutation prevents expression of the mutant RIa allele; however, in the presence of Cre recombinase, excision of

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