Obligaciones y derechos de las partes

Approximately half of all regulators of G protein signaling (RGSs) negatively regulate Gα proteins (Heximer, 2013). In order to do so RGS accelerate GTP hydrolysis at least 40-fold by stabilizing the transition state of the GTPase, which they bind with highest affinity by interaction with all three switch regions of Gα (Berman et al., 1996a, 1996b; Tesmer et al., 1997; Baltoumas et al., 2013) In human 35 RGS proteins are expressed. The RGS domain is the feature common to all of them. This domain is 120-130 amino acids long, composed of a bundle of nine α-helices and can bind directly to the activated Gα subunit (Hollinger and Hepler, 2002; Baltoumas et al., 2013). Of relevance for the present work are the B/R4 and F/RL subfamilies. The B/R4 subfamily consists of RGS1 to 5, 8, 13, 16, 18 and 21 and the F/RL subfamily, also known as RGS-like proteins, includes RhoGEFs (see 4.5), GRKs, AKAPs and sorting nexins (Ross and Wilkie, 2000). Of the F/RL family only the RhoGEFs are relevant for the present study and they were described elsewhere (see 4.5).

The B/R4 RGS proteins can exhibit GAP activity towards Gαiand/or Gαq. Heximer and colleagues found

the GAP activity of RGS2 towards Gαq-stimulated IP3production 5-fold higher than the one of RGS4 and

vice versa RGS4´s GAP activity for Gαi-mediated signaling 8-fold higher than RGS2´s in vitro (Heximer

et al., 1999). The Gαqand Gαiselectivity can be exchanged between RGS2 and 4 by mutation of three

amino acids within the Gα binding pocket of the respective RGS protein into the corresponding amino acids of the other one (Heximer et al., 1999). Nevertheless, RGS2 wild type has GAP activity for Gαiin

membrane-reconstituted system (Cladman and Chidiac, 2002). In 2013, the crystal structure was elucidated for RGS2´s RGS domain in complex with constitutive active Gαq, which lacks its N-terminal

helix (Nance et al., 2013). RGS2 docks to Gαqin an overall similar manner compared to the previously

described RGS Gαi/ocomplexes, but is tilted by seven degrees (Nance et al., 2013). Also this interaction

allows for the conserved mechanism of acceleration of GTPase activity by RGS proteins. If the three amino acids mentioned previously are mutated to the ones in RGS4, the interaction is more similar to the one observed for RGS and Gαi/o. Nevertheless in ventricular myocytes RGS2, 3, 4 and 5 inhibited Gαq

signaling equally well (Hao et al., 2006). Hence, in a signaling pathway specific GAP activity has to depend on additional aspects. One is the type of activated GPCR. Some GPCRs can be recognized by the RGS´ N-terminal region (Zeng et al., 1998; Bernstein et al., 2004; Itoh et al., 2006). As for example RGS5 inhibits Gαqactivation by angiotensin 1 and endothelin ETAreceptor, but not by muscarinic M3receptor

(Zhou et al., 2001). Some RGS proteins work as effector antagonists independent of the GAP activity. In order to do so, they either bind the effector or a region overlapping with the effector binding site at the Gα subunit, thus competing with the effector for binding (Cunningham et al., 2001; Salim et al., 2003; Anger et al., 2004). The latter mechanism was discovered in cells treated with GTPγS. Under this condition RGS2 could not hydrolyze Gαq, but signaling towards PLCβ was still abolished (Cunningham et al.,

2001). Other Gαq effectors are discussed to build high order complexes together with Gα and RGS

binding, but rather inhibit effector association and activation allosterically (Shankaranarayanan et al., 2008; Nance et al., 2013). A part of this study aimed to characterize the allosteric effect of RGS2 on the p63RhoGEF Gαq interaction and investigated downstream signaling in detail (see also 4.5.2.1 PLCβ3

overlaps with p63RhoGEF and RGS2 binding to Gαq). Also Gβγ was suggested to bind RGS proteins

directly, e.g. RGS3, and this interaction was discussed to directly blocked Gβγ signaling (Shi et al., 2001). Additionally direct binding was described between different RGS proteins and mediators downstream of trimeric G proteins (reviewed in (Bansal et al., 2007)).

RGS proteins are tightly regulated

RGS proteins were shown to be regulated by posttranslational modification, translocation as well as changes in expression level:

The palmitoylation at the N-terminus of RGS proteins was discussed to be involved in plasma membrane localization, whereas the lipid modification in the RGS domain are thought to either potentiate or inhibit GAP activity (Hiol et al., 2003; Osterhout et al., 2003; Jones, 2004). In addition phosphorylation can influence the GAP activity positively as well as negatively depending on the RGS protein and kinase involved (Hendriks-Balk et al., 2008).

RGS mRNA levels are regulated tissue as well as receptor specific, for example quick upregulation of RGS2 mRNA was found upon angiotensin II stimulation (Grant et al., 2000; Li et al., 2005). RGS4 mRNA was up and RGS2 mRNA downregulated in models of cardiac hypertrophy (Kach et al., 2012). The protein levels could not be examined directly due to lack of good antibodies. But RGS4 was shown to be ubiquitinated, which probably causes the described relatively short half life of less than an hour (Lee et al., 2005).

Physiology

The first RGS protein described was Sst2p, which inhibits the pheromone-induced mating response in

Saccharomyces cerevisiae and exhibits GAP activity against the yeast Gα, Gpa1 (Dohlman et al.,

1998). In mammals R4 RGS proteins are involved in a wide variety of processes as reviewed elsewhere (Hendriks-Balk et al., 2008). For example RGS2 was shown as a regulator of vascular tone (see 4.7) and was found in almost every tissue investigated in mice and humans (Kehrl and Sinnarajah, 2002). Further mice deficient in RGS2 showed defects in immune response, synapse development and increased anxiety response (Oliveira-dos-Santos et al., 2000). In the vascular pathology of atherosclerosis RGS5 was found downregulated and decreased RGS5 mRNA levels were associated with neointima formation (Geary, 2002; Li et al., 2004; Adams et al., 2006). The expression of RGS1, 13 and 16 in B cells is important for adaptive immune response (Beadling et al., 1999; Han et al., 2006). Almost every RGS protein was found expressed in the mammalian heart and cultured cardiomyocytes, however the expression levels may differ between cell types and regions of the heart (Hendriks-Balk et al., 2008; Zhang and Mende, 2011). For some the expression was really low and others, like RGS2, RGS4 or p115RhoGEF, were highly expressed (Wieland and Mittmann, 2003).