Multilimbed membrane guanylate cyclase signaling system, evolutionary ladder

Abstract

One monumental discovery in the field of cell biology is the establishment of the membrane guanylate cyclase signal transduction system. Decoding its fundamental, molecular, biochemical, and genetic features revolutionized the processes of developing therapies for diseases of endocrinology, cardio-vasculature, and sensory neurons; lastly, it has started to leave its imprints with the atmospheric carbon dioxide. The membrane guanylate cyclase does so via its multi-limbed structure. The inter-netted limbs throughout the central, sympathetic, and parasympathetic systems perform these functions. They generate their common second messenger, cyclic GMP to affect the physiology. This review describes an historical account of their sequential evolutionary development, their structural components and their mechanisms of interaction. The foundational principles were laid down by the discovery of its first limb, the ACTH modulated signaling pathway (the companion monograph). It challenged two general existing dogmas at the time. First, there was the question of the existence of a membrane guanylate cyclase independent from a soluble form that was heme-regulated. Second, the sole known cyclic AMP three-component-transduction system was modulated by GTP-binding proteins, so there was the question of whether a one-component transduction system could exclusively modulate cyclic GMP in response to the polypeptide hormone, ACTH. The present review moves past the first question and narrates the evolution and complexity of the cyclic GMP signaling pathway. Besides ACTH, there are at least five additional limbs. Each embodies a unique modular design to perform a specific physiological function; exemplified by ATP binding and phosphorylation, Ca²⁺-sensor proteins that either increase or decrease cyclic GMP synthesis, co-expression of antithetical Ca²⁺ sensors, GCAP1 and S100B, and modulation by atmospheric carbon dioxide and temperature. The complexity provided by these various manners of operation enables membrane guanylate cyclase to conduct diverse functions, exemplified by the control over cardiovasculature, sensory neurons and, endocrine systems.

Keywords: calcium; carbon dioxide; cardiovascular; cyclic GMP signaling pathways; membrane guanylate cyclase; sensory neurons; surface receptors; transduction modes.

Figure 1

The cyclic AMP and IP3 signaling systems. The cyclic AMP and IP3 signaling systems consist of three separate components: hormone receptor, transducer (G protein) and amplifier adenylate cyclase (for cyclic AMP signaling) or phospholipase C (for IP3 signaling). (Modified from Sharma and Duda, 2014a).

Figure 2

The cyclic GMP signaling system. The cyclic GMP signaling system consists of a single protein. The hormonal signal is recognized by the extracellular receptor domain; the signal is potentiated at the ATP-modulated ARM domain located next to the transmembrane domain in the intracellular portion of the protein (transducer) and the signal is amplified by the cyclase catalytic domain located at the C-terminus of the protein. (Modified from Sharma and Duda, 2014a).

Figure 3

Soluble vs. membrane guanylate cyclase. Upper panel: Graphical representation of soluble and membrane guanylate cyclases. The soluble form is encoded by two genes; it is heterodimeric (subunits α and β) and requires heme for its activity. Both monomers contribute to the catalytic center, but their orientation is unknown yet (Allerston et al., 2013). The membrane guanylate cyclase is encoded by seven genes. It is a single transmembrane spanning protein. The extracellular domain (Ext) is located outside the cell; the transmembrane domain (TM) spans the plasma membrane; and the intracellular domain (ICD) is located inside the cell. The active form is homodimeric. The cyclase catalytic domain is located at the C-terminus of the protein. Both monomers in antiparallel orientation contribute to the catalytic center. Lower panel: Both cyclases are lyases (EC4.6.1.2) and catalyze synthesis of cyclic GMP from GTP. (From: (Sharma and Duda, 2014a).

Figure 4

Heart meets adrenal gland: ANF reciprocal modulation of the two steroidogenic pathways. With every heartbeat, atria stretches and secretes ANF to the circulation. In the adrenal gland, ANF branches and initiates two signaling pathways involving cyclic GMP: one at the fasciculate cells to stimulate the production of cortisol/corticosterone; and the other at glomerular cells to inhibit the production of aldosterone and lower blood pressure.

Figure 5

Model for signal transduction by atrial natriuretic factor-receptor guanylate cyclase (ANF-RGC). (A) Module segments involved in activation. An ANF-signaling element (ANF-SE) resides in the ExtD. A Central Switch (CS), L³⁶⁴, controls the ANF binding site. A binding pocket (BP) is hinged with the CS (van den Akker et al., 2000; Ogawa et al., 2004, 2009). Two disulfide bridged cysteine residues act as a transduction node (TN) to guide the transmembrane migration of the ANF signal to an intracellular, ATP-Regulated Module (ARM; Duda et al., 2005b). Significantly, the TN is active only in the hormone receptor guanylate cyclases but not in the photoreceptor guanylate cyclases (Shahu et al., 2022). ATP amplifies the ANF signal by bringing two critical domains to the surface: a glycine rich cluster G-X-G⁵⁰⁵-X-X-X-G, making surrounding serine and threonine available for phosphorylation (GRC-P) and a 7-aa residue W⁶⁶⁹-TAPELL⁶⁷⁵ motif to activate the core catalytic domain (CCD). (B) Structure of the ARM in its apoform. Four antiparallel β strands and one helix constitute the small lobe. The large lobe is made up of eight α helices and two β strands. The positions of the key G⁵⁰⁵ residue of the GRC motif within the small lobe and of the W⁶⁶⁹-TAPELL⁶⁷⁵ motif within the large lobe are indicated. ATP binding is sandwiched between the two lobes indicated by a star. (C) Activation model for ANF-RGC. Binding of an ANF molecule to the ExtDs of the dimer primes the ANF-SE, by rotating TN. The twisting motion propagates through TM to prepare ARM for ATP binding (Ogawa et al., 2004; Parat et al., 2010). ATP binding triggers a cascade of temporal and spatial changes (Duda et al., 2001c). With G⁵⁰⁵ in GRC-P acting as a pivot, the ATP binding site shifts its position and its floor rotates. There is movement of ARM’s β4 and β5 strands and the loop between them and movement of the αE and F helices that exposes the hydrophobic WTAPELL motif for interaction with CCD (Duda et al., 2009). These structural rearrangements initiate 50% maximal catalytic activity. Full activation is attained after multiple serines and threonines in GRC-P become phosphorylated (Duda et al., 2011b). The conformational changes wrought by ATP binding reduce the affinity of ANF-RGC for ANF and phosphorylation lowers the affinity for ATP binding. Dissociation of ANF and ATP return ANF-RGC to its ground state. (Modified from Duda et al., 2011b; Sharma et al., 2016).

Figure 6

Schematic representation of the luminosity-dependent operation of the ROS-GC-GCAP transduction system. Left panel. An illustration of a typical vertebrate rod. In the DARK a circulating current (arrows) is present. It is outward in the inner segment and carried primarily by K⁺; in the outer segment the net charge is inward, with about 90% of the inward flow carried by the Na⁺ and 10% by Ca²⁺ ions. Na⁺/K⁺ exchange pumps in the inner segment membrane and Na⁺/K⁺-Ca²⁺ exchangers in the outer segment membrane (see also right panels) maintain the overall ionic gradients against the dark flows. The capture of a photon (hν) by a rhodopsin molecule in one of the disc membranes of the outer segment initiates the photo-transduction cascade. Right upper panel, DARK. The components of the Photo-Transduction cascade are shown in the dark/resting steady-state. Cytoplasmic cyclic GMP (red circle), generated by the basal catalytic activity of ROS-GC, keeps a fraction of CNG channels in the plasma membrane open. ROS-GC1 via its ⁴¹⁵M-L⁴⁵⁶ segment is GCAP1-and via ⁹⁶⁵Y-N⁹⁸¹ is GCAP2-bound. Ca²⁺ ions enter the cell via the CNG-channel and are extruded via the Na⁺/K⁺, Ca²⁺-exchanger. Synthesis and hydrolysis of cyclic GMP by ROS-GC and PDE, respectively, occur at a low rate. The heterotrimeric G protein, transducin, is in its GDP-bound state and is inactive. The Ca²⁺ binding proteins calmodulin (CaM), recoverin (Rec) are bound to their target proteins, the CNG-channel, rhodopsin kinase (Rhk), respectively. Right middle panel. Absorption of BRIGHT LIGHT by the visual pigment rhodopsin leads to the activation of the transduction cascade: the GTP-bound α-subunit of transducing activates PDE that rapidly hydrolyzes cyclic GMP. Subsequently, the CNG-channels close and the Ca²⁺-concentration falls. The fall in cytoplasmic [Ca²⁺]i is sensed by Ca²⁺-binding proteins: CaM dissociates from the CNG-channel what leads to an increase in cyclic GMP sensitivity of the channel; recoverin stops inhibiting rhodopsin kinase; rhodopsin becomes phosphorylated. Both Ca²⁺-free GCAPs in their changed configurations activate ROS-GC and synthesis of cyclic GMP increases. Arrestin (Arr) binds to phosphorylated rhodopsin and interferes with the binding and further activation of transducin. Enhancement of cyclic GMP synthesis brings it to its original DARK state level and termination of the cascade, which leads to reopening of CNG channels. Right bottom panel, DIM LIGHT. The initial fall of [Ca²⁺]_i is selectively detected only by GCAP1. In its Ca²⁺-free state GCAP1attains the activated mode and stimulates ROS-GC activity. GCAP2 remains Ca²⁺-bound and in its inhibitory mode. (Reproduced from Sharma and Duda, 2014a).

Figure 7

(A) Modular construction of the ROS-GC1 dimer. A 56 aminoacid leader sequence (LS) precedes the extracellular domain (ExtD) in the nascent, immature protein. All signaling events occur in the intracellular domain (ICD), which is composed of: JMD, juxtamembrane domain; KHD, kinase homology domain; SHD, signaling helix domain; CCD, catalytic core domain; and CTE, C-terminal extension. (B) Interaction with GCAP1 and GCAP2. Two specific switches for Ca²⁺ sensing subunits, one for GCAP1 in the JMD, and one for GCAP2 in the CTE, are located on opposing sides of the CCD. The MGC complex exists as a dimer of homodimers in which two ROS-GC1s combine either two GCAP1s or two GCAP2s.

Figure 8

Independent signaling pathways of ANF/ATP and of NCδ. The trajectory of the ANF pathway (maroon dashed arrow) originates at the ExtD and passes through the TM, ARM and signaling helix domain (SHD) in its course to CCD. In contrast, the trajectory of the NCδ pathway (blue dashed arrow) lies within the CCD. The ANF-RGC dimer is thought to bind a dimer of NCδ, but only a single subunit is shown. Both pathways are the physiological regulators of the mouse blood pressure.

Figure 9

Bicarbonate modulation of ROS-GC activity. (A) Stimulation of ROS-GC in photoreceptor outer segment preparations from WT and neural retina leucine zipper transcription factor knock out (NRL−/−) mice. NRL−/− photoreceptors express ROS-GC1 and GCAP1 exclusively. The dependence of guanylate cyclase activity on bicarbonate is cooperative with an EC₅₀ of 47 mM. The elevated activity at high bicarbonate concentration in WT outer segments is attributed to their additional expression of GCAP2. Error bars show SEM (Duda et al., 2015). (B) Ca²⁺-dependent and-independent modulators of ROS-GC1 activity. Upper panel: three Ca²⁺ sensor proteins – GCAP1, GCAP2, and S100B – and one Ca²⁺-independent modulator, bicarbonate, target individually the indicated domains within the intracellular portion of ROS-GC1. Lower panel: the targeted domains are specific switches all of which signal activation of the catalytic domain. The signaling pathways are indicated as dashed arrows.

Figure 10

Antithetical Ca²⁺ modulation of ROS-GC1 and olfactory neuroepithelial guanylate cyclase (ONE-GC) activities by GCAP1. In the presence of GCAP1, the catalytic activity of recombinant ROS-GC1 decreases as Ca²⁺ is raised from 1 nM to100 mM, but the catalytic activity of recombinant ONE-GC increases. Western blots confirming ROS-GC1 and ONE-GC expression are shown above. Redrawn from Duda et al. (2012a).

Figure 11

Multilimbed membrane guanylate cyclase. In mammals, membrane guanylate cyclase signaling is encoded by seven distinct genes. Through their expression in seven subfamily forms, they perform multi-limbed functions. The surface receptor subfamily, detects hormones and other extracellular chemicals, translate the signal across their plasma membrane domains and by way of their second messenger, cyclic GMP, regulate an intracellular pathway to impact physiology. Sequentially, other similar signaling networks, depicted in the figure, were discovered. Collectively, besides steroidogenesis, they control the physiology of the cardiovasculature, sensory neurons: vision, taste and smell, the intestine and skeletal growth. Uniquely designed, all forms are single-pass transmembrane proteins, embedded with an EXT, TM, CRM and CCD topography. Abbreviations: EXT, extracellular; TM, transmembrane; CRM, catalytic regulatory module; CCD, catalytic core domain.

Figure 12

Ubiquitous expression of MGC signaling systems. MGCs is a multi-switching cyclic GMP generating machine linked with the physiology of cardio-vasculature, smooth muscle relaxation, sensory transduction, neuronal plasticity and memory in mammalian neurosensory, endocrine and peripheral tissues. (Reproduced from Sharma et al., 2015).