G-protein signaling - Rap1A regulation pathway

Rap1A signaling

RAP1A, member of RAS oncogene family (RAP-1A) belongs to a family of small GTP-binding proteins (G-proteins) called monomeric G-proteins. The RAP subfamily consists of four members, RAP-1A, RAP-1B, RAP-2A and RAP-2B proteins [1], [2].

RAP-1A is a target of posttranslational modification via attachment of lipid moieties, such as geranyl, catalyzed by Geranylgeranyltransferase type I (GGTase-I). These posttranslational modifications affect localization and biological activity of RAP-1A [3], [4].

Like other G-proteins, RAP-1A is found in two interconvertible forms, GDP-bound inactive and GTP-bound active forms [5]. Conversion from GDP-bound form to GTP-bound is catalyzed by Guanine nucleotide exchange factors (GEFs). Rap GEF 1 (C3G), Calcium and DAG-regulated GEFs (e.g., RAS guanyl releasing protein 1 (CALDAG-GEFI) and RAS guanyl releasing protein 3 (CalDAG-GEFIII)), Cyclic AMP (cAMP)-dependent GEFs (e.g., Rap GEF 3 (cAMP-GEFI) and Rap GEF 4 (cAMP-GEFII)), Rap GEF 5 (MR-GEF), and RAP1 GTP-GDP dissociation stimulator 1 (Rap1GDS) are known GEFs for RAP-1A. GEF first interacts with the GDP-bound form and releases bound GDP. As a result, a binary complex of the small G protein and GEF is formed. Then GEF in this complex is replaced by GTP resulting in formation of the GTP-bound small G protein.

GEF activity is regulated by an upstream signal. C3G is activated in response to T cell receptor (TCR alpha/beta / CD3 complex) stimulation via Zeta-chain (TCR) associated protein kinase 70kDa (ZAP70)/ v-Crk sarcoma virus CT10 oncogene homolog (avian)-like (CrkL) interaction. C3G interacts with the SH3 domain of CrkL and enhances GEF-activity of C3G [6].

Activity of CALDAG-GEFs and cAMP-GEFs is modulated by the second messengers. Binding of Calcium (Ca(2+)) and Diacylglycerol (DAG) to CALDAG-GEFs activate these GEFs. Cyclic AMP (cAMP) activates cAMP-GEFs. Several signaling pathways, which regulate activity of Adenylate cyclase and Phospholipase C (e.g., PLC-delta), are involved in modulating GEF-activity of CALDAG-GEFs and cAMP-GEFs [7], [8].

Phosphorylation of RAP-1A by cAMP-dependent protein kinase (PKA) leads to activation of RAP-1A [9].

Conversion from GTP-bound active form to GDP-bound inactive form is a result of intrinsic GTPase activity of RAP-1A. This activity is slow, and proteins called GTPase activated proteins (GAPs), e.g., Signal-induced proliferation-associated 1 (SPA1), RAS p21 protein activator 3 (RASA3), RAP1 GTPase activating protein (Rap1GAP), and Tuberous sclerosis 2 (Tuberin), are known to stimulate it [10], [11], [12], [13].

GAP-activity is also regulated by several stimuli. Rap1GAP1 binds specifically to the alpha-subunits of the G(i) family (G-protein alpha-i family) of heterotrimeric G-proteins. Stimulation of the G(i)-coupled receptors translocates Rap1GAP1 from the cytosol to the membrane and decreases the amount of GTP-bound RAP-1A. This decrease in GTP-bound RAP-1A abolishes v-Raf-1 murine leukemia viral oncogene homolog 1 (c-Raf-1) inactivation and activates Mitogen-activated protein kinase (ERK1/2) cascade [12].

RAP-1A binds to c-Raf-1 and competes with RAS proteins for binding to c-Raf-1, thereby antagonizing RAS-dependent activation of c-Raf-1 [14]. On the other hand, v-Raf murine sarcoma viral oncogene homolog B1 (B-Raf), another Raf family member, is activated by RAP-1A [15], [16].

Small G-proteins are also known to cross-talk with each other. RAP-1A modulates activity of RAL and RAS proteins via their respective effectors Ral guanine nucleotide dissociation stimulator (RalGDS) and RAS p21 protein activator 1 (p120GAP) [17], [18]. At the same time, another member of the RAS family, Muscle RAS oncogene homolog (M-Ras), regulates activity of RAP-1A exchange factor MR-GEF [19].

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