Conditionally expressed Gα 15 couples to endogenous receptors in GH 3 cells

Abstract

The mammalian G proteins G15 and G16 couple a wide variety of receptors to phospholipase C (PLC) in co-transfected systems, and it has been suggested that they can be used as tools in agonist-screening systems. Using the reversed tetracycline-controlled transactivation system we generated rat pituitary GH3 cell clones that expressed Galphal5 and Galpha16 conditionally to study the coupling of endogenous receptors to both G proteins. In cells expressing moderate levels of Galpha15, activation of various endogenous receptors increased inositol phosphate production, whereas conditional expression of Galpha16 had no significant effect on agonist-dependent PLC activity. Activation of PLC through Galpha15 in response to carbachol did not increase cytosolic [Ca2+] ([Ca2+]i) but stimulated protein kinase C. While carbachol decreased the secretory activity in non-induced GH3 cells, it increased secretion in cells expressing Galpha15. Our data demonstrate that Galpha15 has a higher functional promiscuity than Galpha16 when studied in a system that preserves physiological G protein and receptor levels. In addition, Galpha15-mediated coupling of a receptor to PLC can change the cellular response to receptor agonists, indicating that downstream cellular functions can be used to detect receptor activation in screening systems employing a promiscuous G protein.

KISS1R: Hallmarks of an Effective Regulator of the Neuroendocrine Axis

“…Kotani et al [29] and Wacker et al [35] demonstrated that KISS1R can also couple to Gα 15/16 and thereby generate inositol trisphosphate (IP3) which mobilizes intracellular Ca 2+ . This finding was not surprising given that Gα 15/16 promiscuously couples a large number of GPCRs to phospholipase Cβ (PLCβ) activation and PIP2 hydrolysis [36,37]. Gα 14 has also been reported to couple some Gα i - and Gα s -coupled receptors to PLCβ activation [38]; however, promiscuous coupling by Gα 14 does not appear to be as widespread as that displayed by Gα 15/16 .…”

The puzzling uniqueness of the heterotrimeric G15 protein and its potential beyond hematopoiesis

“…Exogenous Ga15 expression at physiological levels (promoted by a tetracycline-induced transactivation system) exerted a similar effect by blunting the Ca 2C transient induced through the Gq/11-coupled thyrotropin-releasing hormone receptor (Offermanns et al 2001). G15 expression also inhibited the signaling of b2AR to Gs (Innamorati et al 2009).…”

“…-dependent (Offermanns et al 2001) or independent (PKCm in COS cells unpublished) PKC isoforms triggers several pathways, including those stimulating NFkb (Yang et al 2001), ERK , JNK (Heasley et al 1996) JAK (Lo & Wong 2006, Lo et al 2008, cSrc (Lee et al 2009). Small GTPases, such as Ras and Rho are indirectly modulated by G15, via TPR1 (Yu et al 2008, Su et al 2009) and p63RhoGEF (Yeung & Wong 2009) respectively.…”

“…As a result, the inhibition G15 function reduced lymphocyte activation in response to T cell receptor engagement. In addition, G15 may affect other physiological functions such as transcription, proliferation (Qian et al 1994), differentiation (Ghose et al 1999), secretion (Lippert et al 1997, Offermanns et al 2001. CaM, calmodulin; CaMKII, CaM kinase II; ERK, extracellular signal-regulated kinases; GPCR, G protein coupled receptor; JAK, janus kinase; JNK, cJun NH2-terminal kinase; Lck, leukocyte-specific protein tyrosine kinase; MAPK, mitogen-activated protein kinases; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; MEK, MAPK/ERK kinase; p63, Rho GEF p63 Rho G protein exchange factor; PLC, Phospholipase C; PKC, protein kinase C; PIP2, phosphatidylinositol bisphosphate; Rac1, Rasrelated C3 botulinum toxin substrate 1; Raf-1, V-Raf-1 murine leuk.…”

G Proteins in Drug Screening: From Analysis of Receptor-G Protein Specificity to Manipulation of GPCR-Mediated Signalling Pathways

“…Although Gα15 and Gα16 are clearly more promiscuous than Gα14 [82], they cannot be considered as truly universal [76]. Gα15, in fact, appears to be slightly more promiscuous than Gα16 [32,97]. Interestingly, most of the GPCRs that fail to interact with Gα16 are Gi-selective [for a comprehensive list see refs in 76,98,99].…”