Biochemical, Biophysical and Cellular Techniques to Study the Guanine Nucleotide Exchange Factor, GIV/Girdin

Abstract

Canonical signal transduction via heterotrimeric G proteins is spatiotemporally restricted, i.e. triggered exclusively at the plasma membrane, only by agonist activation of G protein-coupled receptors via a finite process that is terminated within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a non-canonical pathway for activation of heterotrimeric G proteins via the non-receptor guanidine-nucleotide exchange factor, GIV/Girdin. Biochemical, biophysical and functional studies evaluating this pathway have unraveled its unique properties and distinctive spatiotemporal features. As in the case of any new pathway/paradigm, these studies first required an in-depth optimization of tools/techniques and protocols, governed by rationale and fundamentals unique to the pathway, and more specifically to the large multimodular GIV protein. Here we provide the most up-to-date overview of protocols that have generated most of what we know today about non-canonical G protein activation by GIV and its relevance in health and disease.

CCDC88A/GIV promotes HBV replication and progeny secretion via enhancing endosomal trafficking and blocking autophagic degradation

“…The siRNA sequences are shown in Table S3. The sequences of si CCDC88A , si CLTC , si DNM2 , and si HBx were obtained from previous studies [ 23 , 50–52 ], and were synthesized by Sangon Biotech. si GNAI3 (SI00088949), and si RAB5A (SI00301588) were purchased from Qiagen.…”

“…CCDC88A/GIV/Girdin/APE/HkRP1 (coiled-coil domain containing 88A) is the preeminent member of a growing family of proteins, collectively known as guanine nucleotide exchange modulators, that binds Gα-subunits through an evolutionarily conserved short motif [ 21 , 22 ]. CCDC88A can be found in membrane structures of almost all mammalian tissues and various cell lines, and be involved in multiple cellular vesicular trafficking, especially in endosomes and autophagosomes [ 23 ]. CCDC88A activates DNM2 and interacts with R-RAS to mediate endocytosis [ 24 , 25 ].…”

The Gα‐interacting vesicle‐associated protein interacts with and promotes cell surface localization of GRP78 during endoplasmic reticulum stress

“…Samples were separated using standard sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electrotransferred to Immobilon™ FL PVDF membranes (EMD Millipore, Burlington, MA, USA). The recipes for buffers and transfer conditions for GIV were as previously described . Infrared imaging with two‐color detection and quantification of blots were performed according to the manufacturer’s protocols using the Odyssey Fc imaging system (Li‐COR Biosciences).…”

A Circuit for Secretion-coupled Cellular Autonomy in Multicellular Eukaryotes

“…L-glutamine and 5% CO2. Control and GIV shRNA HeLa and Cos7 stable cell lines were selected with 2 μg/ml of Puromycin (GIBCO) using plasmid expressing a shRNA targeting its 3' UTR 123 . Depletion of GIV was verified using GIV-CT antibody with an efficiency of ~95% and cells were extensively validated in prior studies 18,113,114 .…”