Epac2-dependent rap1 activation and the control of islet insulin secretion by glucagon-like peptide-1

Abstract

Glucagon-like peptide-1 (GLP-1) binds its Class II G protein-coupled receptor to stimulate cyclic adenosine monophosphate (cAMP) production and to potentiate the glucose metabolism-dependent secretion of insulin from pancreatic β cells located within the islets of Langerhans. Prior clinical studies demonstrate that this cAMP-mediated action of GLP-1 to potentiate glucose-stimulated insulin secretion (GSIS) is of major therapeutic importance when evaluating the abilities of GLP-1 receptor (GLP-1R) agonists to lower levels of blood glucose in type 2 diabetic subjects. Surprisingly, recent in vitro studies of human or rodent islets of Langerhans provide evidence for the existence of a noncanonical mechanism of β cell cAMP signal transduction, one that may explain how GLP-1R agonists potentiate GSIS. What these studies demonstrate is that a cAMP-regulated guanine nucleotide exchange factor designated as Epac2 couples β cell cAMP production to the protein kinase A-independent stimulation of insulin exocytosis. Provided here is an overview of the Epac2 signal transduction system in β cells, with special emphasis on Rap1, a Ras-related GTPase that is an established target of Epac2.

Figure 10.1

(A) Domain structure of Epac proteins. The two isoforms of Epac are Epac1 and Epac2, each of which is encoded by its own gene. Epac2 is the major isoform of Epac expressed in islets, and it is encoded by the RAPGEF4 gene located at chromosome 2q31–q32. There are three splice variants of Epac2, with Epac2A being the variant expressed in islets. Epac2A has two cAMP-binding domains, a low-affinity site (CNBD-A), important for cellular localization, and a high-affinity site (CNBD-B), important for cAMP-dependent activation of GEF activity. A disheveled, Egl-10, pleckstrin (DEP) domain is responsible for association of Epac2 with intracellular membranes, a Ras exchange motif (REM) domain stabilizes the tertiary structure of the catalytic region, and a Ras association (RA) domain allows the interaction of Epac2 with activated Ras. The CDC25 homology domain (CDC25) catalyzes guanine nucleotide exchange on Rap1, thereby activating it. Epac2B is specifically expressed in the adrenal cortex and lacks the low-affinity cAMP-binding site (CNBD-A). Epac2C is found in the liver and lacks both CNBD-A and DEP domains. All three isoforms have GEF activity to activate Rap1. (B) Role of cAMP in Rap1 activation. Activation of the GLP-1 receptor stimulates G_s, adenylyl cyclase (AC), and cAMP production. The activation of Epac2 is likely to be the major pathway for Rap1 activation in β cells, although PKA can phosphorylate and inactivate Rap1GAP to prolong the activated state of Rap1.

Figure 10.2

Domain structures of PLCε and PKCε. (A) The N-terminal region of PLCε has GEF activity and contains a CDC25 homology domain and possibly a REM domain. The catalytic region contains a pleckstrin homology (PH) domain, EF hand domains, the X and Y boxes, and a core C2 catalytic domain. The C-terminal Ras association (RA) domain contains two Ras association motifs that interact with Ras and Rap. Figure adapted from Bunney and Katan (2006). (B) Illustrated are feedback loops that might be important for sustained activation of Rap1 and PLCε. Rap1 activates PLCε and the GEF domain of PLCε activates Rap1. Note that PLCε-catalyzed hydrolysis of PIP₂ generates DAG and Ca²⁺, both of which activate RasGRP3, thereby catalyzing additional activation of Rap1. (C) Located within the regulatory domain of PKCε is a C1 domain that contains an actin-binding motif and a DAG-binding site. The regulatory domain also contains a C2 domain that binds phospholipids. The kinase domain of PKCε contains a C3 domain, and within it there is an ATP-binding site. Note that phosphorylation of T566 in the activation loop of the C3 domain is essential for PKCε activity. A pseudosubstrate motif at which autophosphorylation occurs is located at the C-terminus of PKCε. Figure adapted from Akita (2002).

Figure 10.3

A model for Epac/Rap1 regulation of K-ATP channels. The K-ATP channel in β cells is a hetero-octamer formed by four SUR1 subunits and four Kir6.2 subunits. SUR1 has 17 transmembrane domains that are grouped into three units (TMD0, TMD1, and TMD2). The intracellular L0 loop between TMD0 and TMD1 interacts with the N-terminal of Kir6.2 (Bryan et al., 2007). Note that Epac2 binds to nucleotide-binding fold-1 (NBF1) of SUR1 and that this interaction may allow cAMP to activate Rap1 and PLCε located within the immediate vicinity of K-ATP channels. Therefore, cAMP is predicted to regulate K-ATP channel activity by stimulating hydrolysis of PIP₂ associated with Kir6.2. Although not shown, it is also possible that the binding of Epac2 to NBF-1 of SUR1 allows Epac2 to allosterically regulate K-ATP channel activity.

Figure 10.4

Regulation of CICR by cAMP. Ca²⁺ influx through VDCCs generates an increase of [Ca²⁺]_i, and this acts as a stimulus for CICR from the ER. The Ca²⁺ release channel that mediates CICR is the ryanodine receptor (RYR). However, Ca²⁺ released from the ER acts as a coagonist with IP₃ to stimulate additional Ca²⁺ release from IP₃ receptor-regulated Ca²⁺ stores (IP3R). Ca²⁺ release via the RYR and IP3R is facilitated by PKA, possibly as a consequence of the phosphorylation of RYR and IP3R. It is also likely that PKA increases the ER Ca²⁺ load by promoting ER Ca²⁺ uptake. Epac2 acts through a pathway involving Rap1, PLCε, PKCε, and CamKII to exert a stimulatory effect at RYR. This action of Epac2 favors additional CICR. Note that Rap1 activation may be stimulated by RasGRP3 since the GEF activity of RasGRP3 is itself stimulated by Ca²⁺ and DAG derived from PLCε-catalyzed hydrolysis of PIP₂. Activated Rap1 may inhibit SERCA, so that leakage of Ca²⁺ from the ER may raise levels of [Ca²⁺]_i and favor CICR. The association of Rap1 with SERCA is inhibited by PKA.

Figure 10.5

CICR regulates insulin secretion. Ca²⁺ influx through VDCCs is the main stimulus for insulin secretion under conditions in which β cells are exposed to elevated levels of glucose. Secretory granules located in an immediate releasable pool (IRP) adjacent to these VDCCs undergo exocytosis in response to the high [Ca²⁺] that exists at the inner mouth of each VDCC. When β cells are exposed to both glucose and GLP-1, exocytosis is amplified due to the fact that CICR is initiated. Amplification of exocytosis occurs because PKA and PKC increase the number of secretory granules within a highly Ca²⁺-sensitive pool (HCSP). These granules are then able to undergo exocytosis in response to CICR that is facilitated by both PKA and Epac2. Ca²⁺ released from the ER also plays a major role in stimulating the mobilization of insulin granules from the reserve pool, an effect mediated by CamKII. The role of Stim1 in β cells is not fully understood but upon store depletion it translocates to the plasma membrane where it may activate store-operated Ca²⁺ entry (SOCE) and also Na⁺ entry through nonselective cation channels. An alternative model suggests that Stim1 activation triggers the formation of a Ca²⁺ influx factor (CIF) that activates iPLA2β, and that iPLA2β regulates SOCE activity. iPLA2β hydrolyzes phospholipids (PL) in the plasma membrane to liberate arachidonic acid (AA) and this lipid metabolite can inhibit Kv2.1 delayed rectifier channels to potentiate membrane depolarization, Ca²⁺ influx, and insulin secretion.

Figure 10.6

(A) Protein interactions that regulate exocytosis. Epac2 stimulates exocytosis by directly interacting with the Ca²⁺ sensor Piccolo (Pic.), Rim2 (Rab GTPase-interacting molecule), and SNAP-25 (SN-25; interaction not shown). Epac2 also indirectly interacts with secretory granule-associated proteins and SNARE apparatus proteins that control exocytosis. These include Rab3A, synaptotagmin (SYTG), VAMP2, and syntaxin (SYTX). Piccolo also interacts with L-type voltage-dependent Ca²⁺ channels (L-VDCC), but the significance of this interaction is not known. (B) Priming of secretory granules by Epac2. In addition to binding plasma membrane SUR1, Epac2 is proposed to bind granular SUR (gSUR) and regulate ClC3 chloride channels to promote granular acidification through the v-type H⁺-ATPase (H⁺ATP). Also shown is the interaction of Munc13-1 with SYTX and Rim2. The C-terminal tail of Rim2 binds weakly to L-VDCC but the significance of this interaction is not known.