RESUMO
The bipartite transcription factor ß-catenin (ß-cat)/T cell factor (TCF), formed by free ß-cat and a given TCF family member, serves as the effector of the developmental Wnt signaling cascade. ß-cat/TCFs also serve as effectors of certain peptide hormones or growth factors during adulthood. We reported that liver-specific expression of dominant-negative Transcription factor 7 like 2 (TCF7L2DN) led to impaired glucose disposal. Here we show that, in this LTCFDN transgenic mouse model, serum and hepatic lipid contents were elevated in male but not in female mice. In hepatocytes, TCF7L2DN adenovirus infection led to stimulated expression of genes that encode lipogenic transcription factors and lipogenic enzymes, while estradiol (E2) treatment attenuated the stimulation, associated with Wnt-target gene activation. Mechanistically, this E2-mediated activation can be attributed to elevated ß-cat Ser675 phosphorylation and TCF expression. In wild-type female mice, ovariectomy (OVX) plus high-fat diet (HFD) challenge impaired glucose disposal and insulin tolerance, associated with increased hepatic lipogenic transcription factor sterol regulatory element-binding protein 1-c (SREBP-1c) expression. In wild-type mice with OVX, E2 reconstitution attenuated HFD-induced metabolic defects. Some of the attenuation effects, including insulin intolerance, elevated liver-weight gain, and hepatic SREBP-1c expression, were not affected by E2 reconstitution in HFD-fed LTCFDN mice with OVX. Finally, the effects of E2 in hepatocytes on ß-cat/TCF activation can be attenuated by the G-protein-coupled estrogen receptor (GPER) antagonist G15. Our study thus expanded the scope of functions of the Wnt pathway effector ß-cat/TCF, as it can also mediate hepatic functions of E2 during adulthood. This study also enriches our mechanistic understanding of gender differences in the risk and pathophysiology of metabolic diseases.
Assuntos
Estradiol/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Via de Sinalização Wnt , beta Catenina/genética , Animais , Benzodioxóis/farmacologia , Dieta Hiperlipídica/efeitos adversos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Glucose/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Resistência à Insulina , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Obesidade/etiologia , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Ovariectomia , Quinolinas/farmacologia , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Fatores Sexuais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , beta Catenina/metabolismoRESUMO
Psoriasis is a common chronic inflammatory skin disease that manifests as scaly erythematous plaques as a consequence of keratinocyte hyperproliferation and inflammation. It is commonly associated with diabetes, obesity, and the metabolic syndrome. While there are numerous approved treatment options available, they have limitations including availability, toxicities such as immunosuppression, and high cost. There is increasing evidence to suggest that several hypoglycemic agents used in the treatment of type 2 diabetes, including glucagon-like peptide-1 receptor agonists, dipeptidyl peptidase-4 inhibitors, thiazolidinediones and biguanides, exert beneficial effects in psoriasis. In this review, we summarize the growing evidence supporting the therapeutic role of hypoglycemic agents in psoriasis and discuss the potential underlying mechanisms. We suggest that dermatologists consider the use of hypoglycemic agents in psoriasis especially in cases with coexisting diabetes and in cases in which immunosuppression is contraindicated. Earlier referral to endocrinology in patients with concomitant diabetes may be appropriate.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Hipoglicemiantes/uso terapêutico , Psoríase/tratamento farmacológico , Biguanidas/farmacologia , Biguanidas/uso terapêutico , Inibidores da Dipeptidil Peptidase IV/farmacologia , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Exenatida , Humanos , Hipoglicemiantes/farmacologia , Liraglutida/farmacologia , Liraglutida/uso terapêutico , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Tiazolidinedionas/farmacologia , Tiazolidinedionas/uso terapêutico , Peçonhas/farmacologia , Peçonhas/uso terapêuticoRESUMO
OBJECTIVE: Disruption of TCF7L2 in mouse pancreatic ß-cells has generated different outcomes in several investigations. Here we aim to clarify role of ß-cell TCF7L2 and Wnt signaling using a functional-knockdown approach. METHODS: Adenovirus-mediated dominant negative TCF7L2 (TCF7L2DN) expression was conducted in Ins-1 cells. The fusion gene in which TCF7L2DN expression is driven by P TRE3G was utilized to generate the transgenic mouse line TCF7L2DN Tet . The double transgenic line was created by mating TCF7L2DN Tet with Ins2-rtTA, designated as ßTCFDN. ß-cell specific TCF7L2DN expression was induced in ßTCFDN by doxycycline feeding. RESULTS: TCF7L2DN expression in Ins-1 cells reduced GSIS, cell proliferation and expression of a battery of genes including incretin receptors and ß-cell transcription factors. Inducing TCF7L2DN expression in ßTCFDN during adulthood or immediately after weaning generated no or very modest metabolic defect, while its expression during embryonic development by doxycycline feeding in pregnant mothers resulted in significant glucose intolerance associated with altered ß-cell gene expression and reduced ß-cell mass. CONCLUSIONS: Our observations support a cell autonomous role for TCF7L2 in pancreatic ß-cells suggested by most, though not all, investigations. ßTCFDN is a novel model for further exploring the role of TCF7L2 in ß-cell genesis and metabolic homeostasis.
RESUMO
Investigations on the metabolic role of the Wnt signaling pathway and hepatic transcription factor 7-like 2 (TCF7L2) have generated opposing views. While some studies demonstrated a repressive effect of TCF7L2 on hepatic gluconeogenesis, a recent study using liver-specific Tcf7l2(-/-) mice suggested the opposite. As a consequence of redundant and bidirectional actions of transcription factor (TCF) molecules and other complexities of the Wnt pathway, knockout of a single Wnt pathway component may not effectively reveal a complete metabolic picture of this pathway. To address this, we generated the liver-specific dominant-negative (DN) TCF7L2 (TCF7L2DN) transgenic mouse model LTCFDN. These mice exhibited progressive impairment in response to pyruvate challenge. Importantly, LTCFDN hepatocytes displayed elevated gluconeogenic gene expression, gluconeogenesis, and loss of Wnt-3a-mediated repression of gluconeogenesis. In C57BL/6 hepatocytes, adenovirus-mediated expression of TCF7L2DN, but not wild-type TCF7L2, increased gluconeogenesis and gluconeogenic gene expression. Our further mechanistic exploration suggests that TCF7L2DN-mediated inhibition of Wnt signaling causes preferential interaction of ß-catenin (ß-cat) with FoxO1 and increased binding of ß-cat/FoxO1 to the Pck1 FoxO binding site, resulting in the stimulation of Pck1 expression and increased gluconeogenesis. Together, our results using TCF7L2DN as a unique tool revealed that the Wnt signaling pathway and its effector ß-cat/TCF serve a beneficial role in suppressing hepatic gluconeogenesis.
Assuntos
Glucose/metabolismo , Fígado/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Animais , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Homeostase/genética , Homeostase/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , beta Catenina/metabolismoRESUMO
p21-activated protein kinase-1 (Pak1) plays a role in insulin secretion and glucagon-like peptide-1 (GLP-1) production. Pak1(-/-) mice were found to carry a defect in ip pyruvate tolerance test (IPPTT), leading us to speculate whether Pak1 represses hepatic gluconeogenesis. We show here that the defect in IPPTT became more severe in aged Pak1(-/-) mice. In primary hepatocytes, 2,2'-dihydroxy-1,1'-dinaphthyldisulfide, a potent inhibitor of group I Paks, reduced basal glucose production (GP), attenuated forskolin- or glucagon-stimulated GP, and attenuated the stimulation of forskolin on the expression of Pck1 and G6pc. In addition, the capacity of primary hepatocytes isolated from Pak1(-/-) mice in GP at the basal level is significantly lower than that of the control littermates. These in vitro observations imply that the direct effect of Paks in hepatocytes is the stimulation of gluconeogenesis and that the impairment in IPPTT in Pak1(-/-) mice is due to the lack of Pak1 elsewhere. Consecutive ip injection of forskolin for 2 weeks increased gut proglucagon expression, associated with improved IPPTT in aged Pak1(-/-) mice and wild-type controls. In addition, administration of the DPP-IV (dipeptidyl peptidase-4) inhibitor sitagliptin for 1 week reversed the defect in IPPTT in aged Pak1(-/-) mice, associated with increased plasma GLP-1 levels. Our observations indicate a potential role of Pak1 in the gut/pancreas/liver axis in controlling glucose disposal and affirmed the therapeutic application of GLP-1 and DPP-IV inhibitors in attenuating hepatic gluconeogenesis.
Assuntos
Glucose/metabolismo , Fígado/metabolismo , Transdução de Sinais , Quinases Ativadas por p21/genética , Animais , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/sangue , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hepatócitos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pirazinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Fosfato de Sitagliptina , Triazóis/farmacologia , Quinases Ativadas por p21/deficiênciaRESUMO
Certain "degradation" products of GLP-1 were found to possess beneficial effects on metabolic homeostasis. Here, we investigated the function of the COOH-terminal fragment of GLP-1, the nonapeptide GLP-1(28-36)amide, in hepatic glucose metabolism. C57BL/6 mice fed a high-fat diet (HFD) for 13 wk were injected intraperitoneally with GLP-1(28-36)amide for 6 wk. A significant reduction in body weight gain in response to HFD feeding was observed in GLP-1(28-36)amide-treated mice. GLP-1(28-36)amide administration moderately improved glucose disposal during glucose tolerance test but more drastically attenuated glucose production during pyruvate tolerance test, which was associated with reduced hepatic expression of the gluconeogenic genes Pck1, G6pc, and Ppargc1a. Mice treated with GLP-1(28-36)amide exhibited increased phosphorylation of PKA targets, including cAMP response element-binding protein (CREB), ATF-1, and ß-catenin. In primary hepatocytes, GLP-1(28-36)amide reduced glucose production and expression of Pck1, G6pc, and Ppargc1a, which was associated with increased cAMP content and PKA target phosphorylation. These effects were attenuated by PKA inhibition. We suggest that GLP-1(28-36)amide represses hepatic gluconeogenesis involving the activation of components of the cAMP/PKA signaling pathway. This study further confirmed that GLP-1(28-36)amide possesses therapeutic potential for diabetes and other metabolic disorders.
Assuntos
Dieta Hiperlipídica , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Gluconeogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Ácido Pirúvico/efeitos adversos , Animais , Peso Corporal/efeitos dos fármacos , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Expressão Gênica/efeitos dos fármacos , Gluconeogênese/genética , Teste de Tolerância a Glucose , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácido Pirúvico/metabolismoRESUMO
Recent studies have demonstrated that the COOH-terminal fragment of the incretin hormone glucagon-like peptide-1 (GLP-1), a nonapeptide GLP-1(28-36)amide, attenuates diabetes and hepatic steatosis in diet-induced obese mice. However, the effect of this nonapeptide in pancreatic ß-cells remains largely unknown. Here, we show that in a streptozotocin-induced mouse diabetes model, GLP-1(28-36)amide improved glucose disposal and increased pancreatic ß-cell mass and ß-cell proliferation. An in vitro investigation revealed that GLP-1(28-36)amide stimulates ß-catenin (ß-cat) Ser(675) phosphorylation in both the clonal INS-1 cell line and rat primary pancreatic islet cells. In INS-1 cells, the stimulation was accompanied by increased nuclear ß-cat content. GLP-1(28-36)amide was also shown to increase cellular cAMP levels, PKA enzymatic activity, and cAMP response element-binding protein (CREB) and cyclic AMP-dependent transcription factor-1 (ATF-1) phosphorylation. Furthermore, GLP-1(28-36)amide treatment enhanced islet insulin secretion and increased the growth of INS-1 cells, which was associated with increased cyclin D1 expression. Finally, PKA inhibition attenuated the effect of GLP-1(28-36)amide on ß-cat Ser(675) phosphorylation and cyclin D1 expression in the INS-1 cell line. We have thus revealed the beneficial effect of GLP-1(28-36)amide in pancreatic ß-cells in vitro and in vivo. Our observations suggest that GLP-1(28-36)amide may exert its effect through the PKA/ß-catenin signaling pathway.
Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Glicemia/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Modelos Animais de Doenças , Desenho de Fármacos , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Técnicas In Vitro , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C beta/metabolismo , Ratos , Transdução de Sinais/fisiologiaRESUMO
The type 2 diabetes risk gene TCF7L2 is the effector of the Wnt signaling pathway. We found previously that in gut endocrine L-cell lines, TCF7L2 controls transcription of the proglucagon gene (gcg), which encodes the incretin hormone glucagon-like peptide-1 (GLP-1). Whereas peripheral GLP-1 stimulates insulin secretion, brain GLP-1 controls energy homeostasis through yet-to-be defined mechanisms. We aim to determine the metabolic effect of a functional knockdown of TCF7L2 by generating transgenic mice that express dominant-negative TCF7L2 (TCF7L2DN) specifically in gcg-expressing cells. The gcg-TCF7L2DN transgenic mice showed reduced gcg expression in their gut and brain, but not in pancreas. Defects in glucose homeostasis were observed in these mice, associated with attenuated plasma insulin levels in response to glucose challenge. The defect in glucose disposal was exacerbated with high-fat diet. Brain Wnt activity and feeding-mediated hypothalamic AMP-activated protein kinase (AMPK) repression in these mice were impaired. Peripheral injection of the cAMP-promoting agent forskolin increased brain ß-cat Ser675 phosphorylation and brain gcg expression and restored feeding-mediated hypothalamic AMPK repression. We conclude that TCF7L2 and Wnt signaling control gut and brain gcg expression and glucose homeostasis and speculate that positive cross-talk between Wnt and GLP-1/cAMP signaling is an underlying mechanism for brain GLP-1 in exerting its metabolic functions.
Assuntos
Encéfalo/metabolismo , Trato Gastrointestinal/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Proglucagon/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Via de Sinalização Wnt , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/agonistas , AMP Cíclico/metabolismo , Trato Gastrointestinal/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Homeostase/efeitos dos fármacos , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Certain single nucleotide polymorphisms (SNPs) in transcription factor 7-like 2 (TCF7L2) are strongly associated with the risk of type 2 diabetes. TCF7L2 and ß-catenin (ß-cat) form the bipartite transcription factor cat/TCF in stimulating Wnt target gene expression. cat/TCF may also mediate the effect of other signaling cascades, including that of cAMP and insulin in cell-type specific manners. As carriers of TCF7L2 type 2 diabetes risk SNPs demonstrated increased hepatic glucose production, we aimed to determine whether TCF7L2 expression is regulated by nutrient availability and whether TCF7L2 and Wnt regulate hepatic gluconeogenesis. We examined hepatic Wnt activity in the TOPGAL transgenic mouse, assessed hepatic TCF7L2 expression in mice upon feeding, determined the effect of insulin on TCF7L2 expression and ß-cat Ser675 phosphorylation, and investigated the effect of Wnt activation and TCF7L2 knockdown on gluconeogenic gene expression and glucose production in hepatocytes. Wnt activity was observed in pericentral hepatocytes in the TOPGAL mouse, whereas TCF7L2 expression was detected in human and mouse hepatocytes. Insulin and feeding stimulated hepatic TCF7L2 expression in vitro and in vivo, respectively. In addition, insulin activated ß-cat Ser675 phosphorylation. Wnt activation by intraperitoneal lithium injection repressed hepatic gluconeogenic gene expression in vivo, whereas lithium or Wnt-3a reduced gluconeogenic gene expression and glucose production in hepatic cells in vitro. Small interfering RNA-mediated TCF7L2 knockdown increased glucose production and gluconeogenic gene expression in cultured hepatocytes. These observations suggest that Wnt signaling and TCF7L2 are negative regulators of hepatic gluconeogenesis, and TCF7L2 is among the downstream effectors of insulin in hepatocytes.
Assuntos
Gluconeogênese/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Inativação Gênica , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Período Pós-Prandial , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/antagonistas & inibidores , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Regulação para Cima/efeitos dos fármacosRESUMO
The Wnt signaling pathway was initially discovered for its role in tumorigenesis and the development of Drosophila and other eukaryotic organisms. The key effector of this pathway, the bipartite transcription factor ß-cat/TCF, is formed by free ß-catenin (ß-cat) and a TCF protein, including TCF7L2. Extensive recent investigations have highlighted the role of the Wnt signaling pathway in metabolic homeostasis and its implication in diabetes and other metabolic diseases. Genome-wide association studies have shown that several key components of the Wnt signaling pathway are implicated in metabolic homeostasis and the development of type 2 diabetes (T2D). Despite controversial observations regarding the role of Wnt signaling in the development and function of pancreatic islets, the discovery of the association between certain single nucleotide polymorphisms of TCF7L2 and T2D susceptibility has fueled great efforts to explore the role of Wnt signaling in the function of pancreatic ß-cells and glucose homeostasis. Here we have introduced our basic understanding of the canonical Wnt signaling pathway, summarized our current knowledge on its implication in metabolic homeostasis and T2D, discussed the work on TCF7L2 as a T2D susceptibility gene, and presented the controversial role of Wnt signaling and TCF7L2 in pancreatic islets as well as their potential metabolic function in other organs. We then expanded our view into the crosstalk among Wnt, insulin and FOXO signaling cascades, which further illustrates the complexity of the Wnt signaling pathway in metabolic homeostasis. Finally, we have presented our perspectives.
RESUMO
Glucose metabolism is tightly controlled by multiple hormones and neurotransmitters in response to nutritional, environmental, and emotional changes. In addition to insulin and glucagon produced by pancreatic islets, two incretin hormones, namely glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP, also known as glucose-dependent insulinotropic peptide), also play important roles in blood glucose homeostasis. The incretin hormones mainly exert their regulatory effects via their corresponding receptors, which are expressed in pancreatic islets as well as many other extra-pancreatic organs. Recent studies have shown that the genes which encode these two incretin hormones can be regulated by the effectors of the Wnt signaling pathway, including TCF7L2, a transcription factor identified recently by extensive genome wide association studies as an important type 2 diabetes risk gene. Interestingly, TCF7L2 and ß-catenin (ß-cat), another effector of Wnt signaling pathway, may also mediate the function of the incretin hormones as well as the expression of their receptors in pancreatic ß-cells. In this review, we have introduced the incretin hormones and the Wnt signaling pathway, summarized recent findings in the field, and provided our perspectives.
RESUMO
Protein transmenembrane (TM) segments participating in helix-helix packing commonly contain small residue patterns (termed GG4 or "small-xxx-small" motifs) at i and i + 4 positions. Within many TM segments - such as the glycophorin A (GpA) sequence L75IxxGVxxGVxxT87- the G17y-xxx-Gly83 motif often occurs in combination with large, usually beta3-branched aliphatic residues at adjacent positions, typified here by Val30 and Val84 residues. To explore the importance of local P-branched character on GpA dimerization, we made systematic replacements to all 16 combinations of single or double Ile, Leu, and AIa residues at GpA TM Val/Val positions 80 and 84. Using the TOXCAT system to assay self-oligomerization in the Escherichia coli inner membrane--we observed that (i) combinations of Val and lie residues maintained, or improved dimerization levels; (ii) single Ala or Leu mutant combinations with Val or Ile maintained near-wild type dimerization affinities; and (iii) in the absence of beta-branching, i.e., Leu/Leu, Ala/Ala and Ala/Leu combinations, GpA dimerization was significantly diminished. An apparent capacity of lle-containing mutants to increase GpA dimerization versus WT likely arises from improved van der Waals packing (vs. Val) within the locus of helix contact, consistent with correlations we noted in lipid accessibility measurements. Examination of several synthetic peptides with sequences corresponding to selected GpA mutants (VV VI, IV II, and LL) confirmed their dimerization on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The overall results reinforce the importance of a beta-branch-containing "ridge" residue to complement a "small-xxx-small groove" in promotion of TM-TM interactions.