Structure

            1.  50kDa

            1.  domain structure (Vanden Heuvel and Peters 2000) check species

                        A/B 1-108

                        C 108-173

                        D 173-249

                        E/F 249-475           

            2.  gene structure

                        a)  human, chromosome 3, 3p25, close to RARb and TRb (at 3p24 and 3p21) (Gearing, Crickmore et al. 1994)-14

                                    1)  spread over 100kb (Zhu, Qi et al. 1995)

                                    2)  gives rise to the three isoforms that differ at their 5’ end due to alternate promoter usage and splicing

                        b)  mouse, chromosome 6, E-F1

                                    1)  spread over 100kb

                        c)  differences in coding exons between PPARg1 and PPARg2

                                    1)  exons 1 through 6 are shared between them

                                    2)  exons A1 and A2 are untranslated exons specific to PPARg1, driven by the P1 promoter

                                    3)  exon B1 is unique to PPARg2, and this encodes an extra 20 amino acids, and is regulated by a separate promoter (P2) which lies >63kb downstream of P1

            3.  RNA transcript

            4.  ligand binding domain

                        a)  ligand binding pockets are three times larger than other nuclear receptors (Nolte, Wisely et al. 1998; Uppenberg, Svensson et al. 1998)

                                    1)  can bind large FAs in multiple conformations (Nolte, Wisely et al. 1998; Uppenberg, Svensson et al. 1998)

                                    1)  PPARg ligand binding domain is over 1000A3 in volume

                                    3)  100A2 whole through which ligands can pass

                        b)  amino acids in the pocket

                                    1)  ligand entry site is composed of amino acid D243 located at the N-terminus of the first b-sheet and amino acids R288, E291, and E295 all located in helix 3

                                    2)  mutation of K319A or L469A prevents ligand binding (Chen, Johnson et al. 2000)

                                    3)  His323 and His449 of PPARg form multiple hydrogen bonds with the ligand (Li, Pascual et al. 2000)-35

                        c)  ligand binding domain is >1300Angstroms (Ricote, Huang et al. 1999)-41

                        d)  substrate interactions with PPARg

                                    1)  PPARg ligands form hydrogen bonds with Y73, H49, and H323 within the ligand binding pocket (Ricote, Huang et al. 1999)-41

            5.  LBD due to ligand binding

                        a)  conformational changes in the LBD especially involving the AF2

                                    1)  rosiglitazone (BRL 49653) make H bonds with Y473 of PPARg and hydrophobic interactions with L469 of PPARg

                                    2)  EPA and GW2433 make H bonds with Y436 on PPARb and hydrophobic interactions with L432

            5.  LXXLL binding domain (Chen, Johnson et al. 2000)

                        a)  L311A, N312A, T316A in helix 4 are necessary for LXXLL binding

            6.  mutagenesis studies by site:

                        P12A hPPARg2 mutant

                                    1)  discovered by (Berger and Moller 2002)-109

                                    2)   frequency of 0.03 to 0.12 in several populations (Berger and Moller 2002)-112

                                    3)  association with obesity in several populations (Berger and Moller 2002)-112

                                    4)  association with lower BMI, improved insulin sensitivity, and reduced incidence of type 2 diabetes (Berger and Moller 2002)-108,113

                                    1)  cannot bind to PPRE, and can’t mediate adipogenesis of PPARg-responsive cells (Bar-Tana 2001)-(Deeb 1998, Masugi 2000)

                                    2)  in patients with a proline at 12, there is a 1.25X increase in diabetic risk (Berger and Moller 2002)-113

                                    3)  little effect on insulin sensitivity (Kersten, Desvergne et al. 2000)

                                    9)  Pro12Pro mutation is associated with increased insulin resistance and increased insulin concentrations when associated with low birth weight (Eriksson 2002 2321)

                                    7)  associated with significantly lower insulin, higher glucose-to-insulin fasting ratio, and lower diastolic blood pressure in African Americans (Kao, Coresh et al. 2003)

                                    8)  greater insulin sensitivity upon mutation in nonobese African Americans (Kao, Coresh et al. 2003)

                        S82

                                    1)  mutagenesis does not stop inhibition of iNOS promoter activity in response to BRL treatment (Li, Pascual et al. 2000)

                                    2)  can still activate the AOX reporter (Li, Pascual et al. 2000)

                        d92

                                    1)   mutagenesis does not stop inhibition of iNOS promoter activity in response to BRL treatment (Li, Pascual et al. 2000)

                        P115Q mutations have little effect on insulin sensitivity (Kersten, Desvergne et al. 2000)

                                    1)  could mediate effects by affecting phosphorylation of Ser114

                                    2)  Gln115 mutant causes greater adipogenesis than wt PPARg when studied in overexpressing cells (Berger and Moller 2002)-110

                        C126A/E127A (Zn2+ finger) – DNA recognition

                                    1)  lowered ability to repress iNOS reporter activity (Li, Pascual et al. 2000)

                                    2)  altered ability to interact with DNA (Li, Pascual et al. 2000)

                                    3)  can still interact with SRC-1 (Li, Pascual et al. 2000)

                        V290M

                                    1)  lower binding affinity for ligand, act as dominant-negatives (Barroso, Gurnell et al. 1999)

                                    2)  severe insulin resistance (Barroso, Gurnell et al. 1999)

                        K301G – critical charge clamp residue

                                    1)  mutagenesis stops activation of AOX reporter construct (Li, Pascual et al. 2000)

                                    2)  no association with SRC633-715 and CBP1-453 (Li, Pascual et al. 2000)

                        H323A

                                    1)  mutagenesis stops activation of an AOX reporter construct (Li, Pascual et al. 2000)

                        F388L

                                    1)  a T to A mutation at nucleotide 1164 is linked to autosomal dominant familial partial lipodystrophy (Hegele, Cao et al. 2002)

                        K442Q (human)

                                    1)  resistant colon cancer lines sometimes have this mutation which prevents their response to PPARg ligands that induce differentiation (Gupta, Sarraf et al. 2003)

                                    2)  transfection with wt PPARg restores the ability of those cells to respond to differentiation induced by PPARg ligands

                                    3)  no defects in heterodimerization or DNA binding, active in reporter assays, but not responsive to growth inhibition in response to treatment with PPARg agonists (Gupta, Sarraf et al. 2003)

                        H449A

                                    1)  mutagenesis has no effect on induction of AOX promoter construct (Li, Pascual et al. 2000)

                        L466A/L467A AF-2

                                    1)  mutation stops BRL49653-mediated induction of PPREx3-TK-LUC (Schulman, Shao et al. 1998)

                                    2)  mutation stops CBP interaction in a M2H approach (Schulman, Shao et al. 1998)

                        P467L mutations

                                    1)  lower binding affinity for ligand, act as dominant-negatives (Barroso, Gurnell et al. 1999)

                                    2)  severe insulin resistance (Barroso, Gurnell et al. 1999)

                        E469 – critical charge clamp residue

                                    1)  mutagenesis stops activation of AOX reporter construct (Li, Pascual et al. 2000)

                                    2)  no association with SRC633-715 and CBP1-453 (Li, Pascual et al. 2000)

                        K319A or L469A

                                    1)  mutation of K319A or L469A prevents ligand binding (Chen, Johnson et al. 2000)

                        L468A/E471A  - acts as a dominant negative forms

                                    1)  PPARg L468A/E471A can bind to ligand and DNA but not to coactivators (Wang, Fu et al. 2001)-26

                        DBD

                                    1)  loss of the DBD prevents inhibition of iNOS reporter activity (Li, Pascual et al. 2000)

                                    2)  can still interact with CBP in the presence of ligand, but cannot interact with SRC (Li, Pascual et al. 2000)

                        AF2

                                    1)  mutagenesis stops activation of AOX reporter construct (Li, Pascual et al. 2000)

                                    2)  no association with SRC (Li, Pascual et al. 2000)

            7.  hybrid construction:  PPARd/g fusion construct

                        a)  amino-terminal half of PPARb substituted with the N terminal part of PPARg is able to respond to PPARb ligands and act like a PPARg receptor (Brun and Spiegelman 1997)