Asymmetric divisions are crucial for generating cell diversity; they rely on coupling between polarity cues and spindle positioning, but how this coupling is achieved is poorly understood. In one-cell stage Caenorhabditis elegans embryos, polarity cues set by the PAR proteins mediate asymmetric spindle positioning by governing an imbalance of net pulling forces acting on spindle poles. We found that the GoLoco-containing proteins GPR-1 and GPR-2, as well as the Galpha subunits GOA-1 and GPA-16, were essential for generation of proper pulling forces. GPR-1/2 interacted with guanosine diphosphate-bound GOA-1 and were enriched on the posterior cortex in a par-3- and par-2-dependent manner. Thus, the extent of net pulling forces may depend on cortical Galpha activity, which is regulated by anterior-posterior polarity cues through GPR-1/2.

Patients with human papillomavirus associated (HPV+) head and neck cancer (HNC) demonstrate significantly improved survival outcome compared to those with HPV− negative (HPV−) tumors. Published data examining this difference offers conflicting results to date. We systematically investigated the radiation sensitivity of all available validated HPV+ HNC cell lines and a series of HPV− HNC cell lines using in vitro and in vivo techniques. HPV+ HNCs exhibited greater intrinsic radiation sensitivity (average SF2 HPV− 0.59 vs. HPV+ 0.22, p<0.0001), corresponding with a prolonged G2/M cell cycle arrest and increased apoptosis following radiation exposure (percent change 0% vs. 85%, p=0.002). A genome-wide microarray was used to compare gene-expression 24 hours following radiation between HPV+ and HPV− cell lines. Multiple genes in TP53 pathway were upregulated in HPV+ cells (Z score 4.90), including a 4.6 fold increase in TP53 (p<0.0001). Using immortalized human tonsillar epithelial cells, increased radiation sensitivity was seen in cell expressing HPV-16 E6 despite the effect of E6 to degrade p53. This suggested that low levels of normally functioning p53 in HPV+ HNC cells could be activated by radiation, leading to cell death. Consistent with this, more complete knockdown of TP53 by siRNA resulted in radiation resistance. These results provide clear evidence, and a supporting mechanism, for increased radiation sensitivity in HPV+ HNC relative to HPV− HNC. This issue is under active investigation in a series of clinical trials attempting to de-escalate radiation (and chemotherapy) in selected patients with HPV+ HNC in light of their favorable overall survival outcome.

Abstract. Heterotrimeric G-proteins are intracellular partners of G-protein-coupled receptors (GPCRs). GPCRs act on inactive Ga·GDP/Gbg heterotrimers to promote GDP release and GTP binding, resulting in liberation of Ga from Gbg. Ga·GTP and Gbg target effectors including adenylyl cyclases, phospholipases and ion channels. Signaling is terminated by intrinsic GTPase activity of Ga and heterotrimer reformation -a cycle accelerated by 'regulators of G-protein signaling' (RGS proteins). Recent studies have identified several unconventional G-protein signaling pathways that diverge from this CMLS, Cell. Mol. Life Sci. 62 (2005) 551-577 1420-682X/05/050551-27 DOI 10.1007/s00018-004-4462-3 © Birkhäuser Verlag, Basel, 2005 CMLS Cellular and Molecular Life Sciencesstandard model. Whereas phospholipase C (PLC) b is activated by Ga q and Gbg, novel PLC isoforms are regulated by both heterotrimeric and Ras-superfamily G-proteins. An Arabidopsis protein has been discovered containing both GPCR and RGS domains within the same protein. Most surprisingly, a receptor-independent Ga nucleotide cycle that regulates cell division has been delineated in both Caenorhabditis elegans and Drosophila melanogaster. Here, we revisit classical heterotrimeric G-protein signaling and explore these new, non-canonical G-protein signaling pathways.

The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein ␣ subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G␣ i/o -Loco (GoLoco) motif predicted to represent a second G␣ interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G␣ i1 , G␣ i2 , and G␣ i3 in their GDPbound forms. In GTP␥S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G␣ i1 . Both regions also stabilize G␣ i1 in its GDPbound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF 4 Ϫ . Our results indicate that both RGS12 and RGS14 harbor two distinctly different G␣ interaction sites: a previously recognized N-terminal RGS box possessing G␣ i/o GAP activity and a C-terminal GoLoco region exhibiting G␣ i GDI activity. The presence of two, independent G␣ interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G␣ GAP activity.In the standard model of heterotrimeric G-protein signaling, cell surface receptors (GPCRs) 1 are coupled to a membraneassociated heterotrimer composed of G␣, G␤, and G␥ subunits (1, 2). G␤ and G␥ form an obligate heterodimer that binds tightly to GDP-bound G␣ subunits, enhancing G␣ coupling to receptor and inhibiting its release of GDP (i.e. G␤␥ dimers exhibit "guanine nucleotide dissociation inhibitor" (GDI) activity; Refs. 3-5). Upon agonist binding, the GPCR becomes a guanine nucleotide exchange factor (GEF) and promotes replacement of bound GDP for GTP on the G␣ subunit. The binding of GTP changes the conformation of three "switch" regions within G␣, allowing G␤␥ dissociation. GTP-bound G␣ and free G␤␥ subunits both initiate signals by interactions with downstream effector proteins until the intrinsic guanosine triphosphatase (GTPase) activity of G␣ returns the protein to the GDP-bound state. Reassociation of G␤␥ with GDP-bound G␣ obscures critical effector contact sites and terminates all effector interactions (6, 7). Hence, the duration of heterotrimeric G-protein signaling is controlled by the guanine nucleotide state of the G␣ subunit.We and others have identified a family of GTPase-activating proteins (GAPs) for G␣ subunits, the "regulators of G-protein signaling" or RGS proteins (8 -11). These proteins all contain a hallmark "RGS box," which accelerates the intrinsic GTPase rate of G␣ subunits by binding avidly to the transition state for GTP hydrolysis (12). Discovery of RGS box-mediated GAP activity finally resolved the paradox that GPCR-stimulated signals terminate much faster in vivo than predicted from the slow GTP hydrolysis rates exhibited by purified G␣ subunits in vitro (13). However, R...

The GoLoco motif is a 19-amino-acid sequence with guanine nucleotide dissociation inhibitor activity against G-alpha subunits of the adenylyl-cyclase-inhibitory subclass. The GoLoco motif is present as an independent element within multidomain signaling regulators, such as Loco, RGS12, RGS14, and Rap1GAP, as well as in tandem arrays in proteins, such as AGS3, G18, LGN, Pcp-2/L7, and Partner of Inscuteable (Pins/Rapsynoid). Here we discuss the biochemical mechanisms of GoLoco motif action on G-alpha subunits in light of the recent crystal structure of G-alpha-i1 bound to the RGS14 GoLoco motif. Currently, there is sparse evidence for GoLoco motif regulation of canonical G-protein-coupled receptor signaling. Rather, studies of asymmetric cell division in Drosophila and Caenorhabditis elegans, as well as mammalian mitosis, implicate GoLoco proteins, such as Pins, GPR-1/GPR-2, LGN, and RGS14, in mitotic spindle organization and force generation. We discuss potential mechanisms by which GoLoco/Galpha complexes might modulate spindle dynamics.

The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical problem. In this study we show that overexpression of the oncogenic receptor kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated and tightly associated with EGFR expression in cells resistant to cetuximab (CtxR cells). Using RNAi methods and novel AXL targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation and MAPK signaling in CtxR cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in CtxR cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft assays, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL targeting drugs to treat cetuximab-resistant cancers.

Young patients with head and neck squamous cell carcinoma (HNSCC) appear to have unique clinical profiles based on history of alcohol and tobacco abuse.

Human papillomavirus (HPV), a known etiology of a subset of head and neck squamous cell carcinomas (HNCs), causes numerous alterations in normal cellular functions. This article reviews the biology, detection and treatment of HPV-positive HNC. The role of HPV oncoproteins in tumor development, the natural history of HPV infection, and risk factors for and prevention of transmission of oral HPV are considered. Commonly used methods for detecting HPV infection including limitations of these methods are discussed to aid the practicing clinician in utilizing these tests in their clinical practice. Clinical characteristics of HPV-positive HNC including potential explanations for the improved outcomes seen in patients with HPV-positive HNC are assessed. Ongoing clinical trials specific for patients with HPV-positive HNC are described and areas in need of additional research are summarized. Until the results of ongoing trials are known, treatment of HPV-positive HNC should not differ in clinical practice from treatment of similar non-HPV related cancers.