Single nucleotide polymorphisms (SNPs) within the ADCY5 gene, encoding adenylate cyclase 5, are associated with elevated fasting glucose and increased type 2 diabetes (T2D) risk. Despite this, the mechanisms underlying the effects of these polymorphic variants at the level of pancreatic β-cells remain unclear. Here, we show firstly that ADCY5 mRNA expression in islets is lowered by the possession of risk alleles at rs11708067. Next, we demonstrate that ADCY5 is indispensable for coupling glucose, but not GLP-1, to insulin secretion in human islets. Assessed by in situ imaging of recombinant probes, ADCY5 silencing impaired glucose-induced cAMP increases and blocked glucose metabolism toward ATP at concentrations of the sugar >8 mmol/L. However, calcium transient generation and functional connectivity between individual human β-cells were sharply inhibited at all glucose concentrations tested, implying additional, metabolism-independent roles for ADCY5. In contrast, calcium rises were unaffected in ADCY5-depleted islets exposed to GLP-1. Alterations in β-cell ADCY5 expression and impaired glucose signaling thus provide a likely route through which ADCY5 gene polymorphisms influence fasting glucose levels and T2D risk, while exerting more minor effects on incretin action.

A signaling complex enables the compartmentalized regulation of cyclic AMP signaling by calcium entering through a specific channel.

Protein kinase A anchoring proteins (AKAPs) provide the backbone for targeted multimolecular signaling complexes that serve to localize the activities of cAMP. Evidence is accumulating of direct associations between AKAPs and specific adenylyl cyclase (AC) isoforms to facilitate the actions of protein kinase A on cAMP production. It happens that some of the AC isoforms (AC1 and AC5/6) that bind specific AKAPs are regulated by submicromolar shifts in intracellular Ca2+. However, whether AKAPs play a role in the control of AC activity by Ca2+ is unknown. Using a combination of co-immunoprecipitation and high resolution live cell imaging techniques, we reveal an association of the Ca2+-stimulable AC8 with AKAP79/150 that limits the sensitivity of AC8 to intracellular Ca2+ events. This functional interaction between AKAP79/150 and AC8 was observed in HEK293 cells overexpressing the two signaling molecules. Similar findings were made in pancreatic insulin-secreting cells and cultured hippocampal neurons that endogenously express AKAP79/150 and AC8, which suggests important physiological implications for this protein-protein interaction with respect to Ca2+-stimulated cAMP production.

-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC␥2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC␥2 and corresponding PLC␥1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5-and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

Rho family GTPases are important cellular switches and control a number of physiological functions. Understanding the molecular basis of interaction of these GTPases with their effectors is crucial in understanding their functions in the cell. Here we present the crystal structure of the complex of Rac2 bound to the split pleckstrin homology (spPH) domain of phospholipase C-gamma(2) (PLCgamma(2)). Based on this structure, we illustrate distinct requirements for PLCgamma(2) activation by Rac and EGF and generate Rac effector mutants that specifically block activation of PLCgamma(2), but not the related PLCbeta(2) isoform. Furthermore, in addition to the complex, we report the crystal structures of free spPH and Rac2 bound to GDP and GTPgammaS. These structures illustrate a mechanism of conformational switches that accompany formation of signaling active complexes and highlight the role of effector binding as a common feature of Rac and Cdc42 interactions with a variety of effectors.

Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLC␥ isoforms, only PLC␥ 2 has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLC␥ 2 required for activation. Based on reconstitution experiments with isolated PLC␥ variants and Rac2, we show that an unusual pleckstrin homology (PH) domain, designated as the split PH domain (spPH), is both necessary and sufficient to effect activation of PLC␥ 2 by Rac2. We also demonstrate that Rac2 directly binds to PLC␥ 2 as well as to the isolated spPH of this isoform. Furthermore, through the use of NMR spectroscopy and mutational analysis, we determine the structure of spPH, define the structural features of spPH required for Rac interaction, and identify critical amino acid residues at the interaction interface. We further discuss parallels and differences between PLC␥ 1 and PLC␥ 2 and the implications of our findings for their respective signaling roles.Phosphoinositide-specific phospholipase C (PLC) 3 enzymes have been established as crucial signaling nodes involved in regulation of a variety of cellular functions via hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate. There are six major families of PLC enzymes (PLC␤, -␥, -␦, -⑀, -, and -) that share a common core of domains related to catalysis and are distinguished by family-specific regulatory regions (1-3). The two isoforms of the PLC␥ family, PLC␥ 1 and PLC␥ 2 , uniquely incorporate an array of domains comprising two SH2 domains, an SH3 domain, and an internal or "split" PH domain (spPH). spPHs represent a unique subclass of PH domains that are characterized by insertions of one or several autonomously folded protein modules encoded within the boundaries of PH domain sequences (4). This array also contains sites for phosphorylation by several receptor (e.g. epidermal growth factor and platelet-derived growth factor receptors) and nonreceptor tyrosine kinases. In addition to tyrosine phosphorylation, multiple protein-protein interactions (mainly mediated by SH2 and SH3 domains) contribute to PLC␥ activation and have an important role in localizing the enzyme to protein complexes in different cellular compartments (5, 6). However, the elucidation at the molecular level of how PLC␥ isoforms are regulated remains an area of intense study.Despite the common domain organization shared by the PLC␥ 1 and PLC␥ 2 isoforms, studies using gene-targeting approaches demonstrated that each has a distinct biological role (7,8). Different functions of PLC␥ 1 (essential role in embryonic development) and PLC␥ 2 (requirement for development and function of hematopoietic cells) to some degree reflect their different expression patterns and, in particular, the abundance of PLC␥ 2 in hematopoietic cells. However, studies of different cell types where both isoforms are present (e.g. platelets, macrophages/mo...

Here we describe an improved sensor with reduced pH sensitivity tethered to adenylyl cyclase (AC) 8. The sensor was used to study cAMP dynamics in the AC8 microdomain of MIN6 cells, a pancreatic β-cell line. In these cells, AC8 was activated by Ca2+ entry through L-type voltage-gated channels following depolarisation. This activation could be reconstituted in HEK293 cells co-expressing AC8 and either the α1C or α1D subunit of L-type voltage-gated Ca2+ channels. The development of this improved sensor opens the door to the study of cAMP microdomains in excitable cells that have previously been challenging due to the sensitivity of fluorescent proteins to pH changes.

SummaryAdenylyl cyclase (AC) isoforms can participate in multimolecular signalling complexes incorporating A-kinase anchoring proteins (AKAPs). We recently identified a direct interaction between Ca 2+ -sensitive AC8 and plasma membrane-targeted AKAP79/150 (in cultured pancreatic insulin-secreting cells and hippocampal neurons), which attenuated the stimulation of AC8 by Ca 2+ entry (Willoughby et al., 2010). Here, we reveal that AKAP79 recruits cAMP-dependent protein kinase (PKA) to mediate the regulatory effects of AKAP79 on AC8 activity. Modulation by PKA is a novel means of AC8 regulation, which may modulate or apply negative feedback to the stimulation of AC8 by Ca 2+ entry. We show that the actions of PKA are not mediated indirectly via PKA-dependent activation of protein phosphatase 2A (PP2A) B56d subunits that associate with the N-terminus of AC8. By site-directed mutagenesis we identify Ser-112 as an essential residue for direct PKA phosphorylation of AC8 (Ser-112 lies within the N-terminus of AC8, close to the site of AKAP79 association). During a series of experimentally imposed Ca 2+ oscillations, AKAP79-targeted PKA reduced the on-rate of cAMP production in wild-type but not non-phosphorylatable mutants of AC8, which suggests that the protein-protein interaction may provide a feedback mechanism to dampen the downstream consequences of AC8 activation evoked by bursts of Ca 2+ activity. This finetuning of Ca 2+ -dependent cAMP dynamics by targeted PKA could be highly significant for cellular events that depend on the interplay of Ca 2+ and cAMP, such as pulsatile hormone secretion and memory formation.