생명과학회지 (Journal of Life Science)

한국생명과학회 (Korean Society of Life Science)
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Chromophore 형성과 rhodopsin kinase 활성을 이용한 항활성 로돕신 mutant의 분석

Chromophore formation and phosphorylation analysis of constitutively active rhodopsin mutants

  • 발행 : 2007.06.25
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초록

G protein-coupled receptor, (GPCR)는 세포외부의 신호를 인식 시 G 단백질을 활성화시켜 신호를 전달하며 kinase에 의한 인산화를 통하여 지속적인 신호전달을 억제한다. 외부 신호물질이 없는 조건에서도 활성을 나타내는 항활성 돌연변이종(CAM)은 GPCR의 신호전달 이상에 기인한 질병 치료나 활성화 구조변화의 좋은 연구대상이다. 희미한 빛을 인식하는 시각수용체인 로돕신의 CAM으로는 salt bridge에 직접적인 영향을 미치는 돌연변이인 G90D, El13Q, 그리고 K296E와, 직접적인 영향이 없는 돌연변이인 E134q와 M25Y등 두 가지 계통의 종류가 알려져 있다. 본 연구에서는 각각의 돌연변이가 복합된 mutant를 구성하여 agonist와 inverse agonist에 대한친화도와 로돕신 kinase에 대한 활성을 조사하여 각 종에서의 구조변화의 차이를 분석하였다. 로돕신 mutant의constitutive activity는 all-trans-retinal에 대한 친화도에 비례하며 11-cis-retinal에 대한 친화도와는 역상관 관계를 보여준다. 같은 계통에 속하는 돌연변이가 합쳐진 복합 mutant는 단일 mutant에 비하여 미약한 정도의 로돕신 kinase 항활성화 증가를 보여주나, 다른 계통에 속하는 두 가지 돌연변이가 합쳐진 mutant는 항활성화가 크게 증가되었음을 보여주었다. 이 결과는 다른 계통에 속하는 mutant에서는 상이한 구조변화가 일어나며 로돕신이완전한 활성화에 이르기 위해서는 최소한 두 가지 종류의 돌연변이에 의하여 생기는 구조변화들이 함께 일어나야함을 의 미 한다. G protein 활성화와 유사한 항활성화 분석 결과는 rhodopsin kinase가 인식하는 로돕신의 활성화상태 구조가 G protein이 인식하는 구조와 유사함을 의미한다. 특히 가장 강한 활성을 나타내는 El13Q/E134Q/M257Y는 활성화상태 GPCR 단백질의 결정 시도에 이용 될 수 있을 것이다.

G protein coupled receptors (GPCRs) transmit various extracellular signals into the cells. Upon binding of the ligands, conformational changes in the extracellular and/or transmembrane (TM) domains of CPCRs were propagated into the cytoplasmic (CP) domain of the molecule leading to the activation of their cognate heterotrimeric C proteins and kinases. Constitutively active GPCR mutants causing the activation of C Protein signaling even in the absence of ligand binding are of interest for the study of activation mechanism of GPCRs. Two classes of constitutively active mutations, categorized by their effects on the salt bridge between Ell3 and K296, were found in the TM domain of rhodopsin. Opsin mutants containing combinations of the mutations were constructed to study the conformational changes required for the activation of rhodopsin. Rhodopsin chromophore regenerated with 11-cis-retinal showed a thermal stability inversely correlated with its constitutive activity. In contrast, rhodopsin mutants exhibited a binding affinity to an agonist, all-trans-retinal, in a constitutive activity-dependent manner. In order to test whether the conformational changes responsible for the activation of trans-ducin (Gt) are the same as the conformation required for the recognition of rhodopsin kinase, analysis of the mutants were carried out with phosphorylation by rhodopsin kinase. Rhodopsin mutants containing combinations of different classes of the mutations showed a strong synergistic effect on the phosphorylation of the mutants in the dark as similar to that of Gt activation. The results suggest that at least two or three kinds of segmental and independent conformational changes are required for the activation of rhodopsin and the conformational changes responsible for activating rhodopsin kinase and Gt are similar to each other.

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