This chapter has focused on the examination of the coupling of G proteins to the somatostatin receptor SSTR1. With the molecular cloning of five distinct subtypes, analysis of each individual receptor is possible. It is now apparent that in many tissues and cells more than one subtype is expressed, making it difficult to assign G proteins and effectors to individual receptor subtypes. Expression of individual receptor subtypes in cell lines that have little or no other somatostatin receptor provides a system for the analysis of specific receptor-G protein coupling that is easier to interpret.
This chapter discusses the cross-linking of synaptoneurosome G proteins. A means of probing the organizational structure of G proteins in their native membrane environment is to examine the structures obtained after treatment of the membranes with cross-linking agents. Greater the stringency of the cross-linking reactions, the more credible the interpretations of the cross-linked structures is established. For this purpose a sulfhydryl-based monofunctional cross-linker, p-phenylenedimaleimide (p-PDM) is appropriate both because of the short phenyl group as spacer between the maleimide or sulfhydryl reactive groups and because it is known that G-proteins contain multiple sulfhydryl residues for potential cross-linking. Synaptoneurosome membranes are employed because they contain essentially all of the known species of G-proteins and are a relatively specific type of membrane from the brain cortex. Detergents exert varying effects on the organizational structures of G proteins. For example, extraction of membranes with Lubrol yields heterotrimeric structures (monomers) of G proteins whereas extraction with digitonin yields large, presumably multimeric structures. Cross-linking of Lubrol and digitonin extracts demonstrates this point.
This chapter describes the methods that have been used for making monoclonal antibodies against G protein subunits. Monoclonal antibodies can be generated successfully using either G proteins purified from mammalian tissues or recombinant G proteins purified from bacterial or eukaryotic expression systems. To make α-subunit-specific antibodies, either the heterotrimer (αβγ) or the α-subunit monomer, as the antigen is used. Because G proteins belong to a superfamily of proteins that share high sequence homology along with varying among species, both the subtype specificity and the species selectivity of monoclonal antibodies to G proteins need to be determined. As monoclonal antibodies generated using purified G proteins rather than synthetic peptides react with an unknown amino acid sequence or three-dimensional structure, identification of the epitope is useful in the application of the antibody. In addition, the reactivity of these monoclonal antibodies to native versus denatured proteins, heterotrimers versus monomers, GDP-bound versus GTP-bound, and ADP-ribosylated or lipid-modified G proteins can be identified to facilitate their use.
This chapter reviews the understanding of G protein expression and function in α1-receptor response pathways in cardiac tissue. In particular, the evidence that establishes the importance of sympathetic innervation in the functional acquisition of a pertussis toxin-sensitive G protein linking the α1-adrenergic receptor to inhibition of automaticity in the mature, innervated heart is of importance. The effects of α1-agonists to modulate contractile function are discussed within the context of the current understanding of α1-adrenergic receptor subtypes and their linkage to G proteins in cardiac myocytes. From the rather correlative nature of the data available, it will become apparent that despite the early identification of a functional linkage between a α1-receptor subtype, a pertussis toxin-sensitive G protein, and an inhibitory chronotropic response, the molecular identity and/or the precise biochemical events that control α1-receptor signaling through this pathway have yet to be uncovered. The G proteins represent a fundamental and widespread mechanism for the transmission of signals from agonist-occupied cell surface receptors to a number of membrane-bound effector mechanisms, including enzymes (ade nylylcyclases and phospholipases) and ion channels.
Low oxygen availability in the high altitude milieu causes adverse physiological and pathological consequences to the cardiopulmonary system. A key role is played by proteins in maintaining optimal cardiac function under stress. Differential response to hypoxia may be linked to the susceptibility of proteins to free radical induced modifications. The present study was designed to understand the significance of protein oxidation and ER stress in the myocardial response to hostile environments.Sprague–Dawley rats were exposed to simulated hypoxia equivalent to 223 mm Hg pressure, screened on the basis of time taken for onset of a characteristic hyperventilatory response and categorized as susceptible (< 10 min), normal (10–25 min) or tolerant (> 25 min). Protein modifications and activity of cellular proteolytic enzymes were assayed in myocardial tissue extracts to identify alterations in protein homeostasis. To evaluate the ER stress response, expression of various ER marker chaperones was investigated.Susceptible animals displayed a distinct increase in protein oxidation and intracellular thiol content. They showed higher expression of ER stress hallmarks, GRP78, PDI and ERO1α, and exhibited a greater activation of the proteasome and calpain proteolytic systems, associated with elevated oxidized proteins. While a marked upregulation in the prosurvival signaling cascade PI3K/Akt/mTOR was observed in tolerant animals, the expression of pro-apoptotic caspase-3 and CHOP remained unaltered.Thus, higher susceptibility to hypoxia is linked to a disruption in the proteostasis and activation of the ER stress response. Enhanced tolerance to hostile environments may be contributed by better maintenance of protein folding homeostasis.
This chapter discusses methods to examine the effects that structural changes in the parathyroid hormone and parathyroid hormone-related protein (PTH/PTHrP) receptor, such as tail truncation and mutations in intracellular loops, have on the signal transduction through both cyclic AMP-and intracellular Ca2+-dependent pathways. The Xenopus oocyte expression system, therefore, provides some evidence as to the location of interaction sites in the receptor for the G proteins that signal through adenylate cyclase and PLC activation. Because of the relative ease of measuring both adenylate cyclase and phospholipase C (PLC) activation in Xenopus oocytes, this System is used to examine the effects of specific mutations on the functional properties of the PTH/PTHrP receptor. The injection of exogenous mRNAs encoding receptors or ion channels in oocytes will often lead to the synthesis of full-length proteins, which may then undergo posttranslational processing. Such proteins are then inserted into the plasma membrane in a functional form and can be screened for using an assay of receptor or ion channel function. The properties of oocytes, together with the tools of receptor mutagenesis and chimeric protein construction, have enabled the dissection of the functional roles of specific regions of receptors, ion channels, and other proteins using this system.
This chapter focuses on methods utilizing G protein subunit antibodies and receptor sequence peptides to probe α2AR-G protein interactions. Activation of the α2AR inhibits adenylylcyclase through the activation of a pertussis toxin-sensitive guanine nucleotide-binding protein Gi (22). As all three Gi subtypes, along with Go, are inactivated by pertussis toxin, a method to discriminate among the Gi subtypes is desirable in order to determine which G protein(s) are essential for α2AR inhibition of adenylylcyclase. Use of antisera directed against the C-terminal region of Gil/Gi2 or Gi3 proteins in a high-affinity α2AR agonist-binding assay and adenylylcyclase assay demonstrated that both Gi2 and Gi3 transduce α2AR inhibition of adenylylcyclase. Various approaches are applicable to examining pertussis toxin-sensitive receptor signal transduction, are discussed. The G-protein-coupled receptors, including the α2AR, have a similar topographical arrangement in the membrane. These receptors contain seven membrane-spanning domains connected by three intracellular and three extracellular loops, with the C terminus intracellular.
