Zhu S J, Toews M L
Department of Pharmacology, University of Nebraska Medical Center, Omaha 68198-6260.
Mol Pharmacol. 1994 Feb;45(2):255-61.
During the course of equilibrium competition binding assays with intact cells, agonists induce conversion of beta-adrenergic receptors (BARs) from a native form with high affinity for agonists to a form with a markedly lower apparent affinity. The roles of receptor internalization, receptor-Gs coupling, and receptor phosphorylation in this agonist-induced conversion to the low affinity form were investigated. Agonist and antagonist competition for [125I]iodopindolol binding to intact cells was measured in mouse L cells expressing wild-type BARs (C+I+), mutated BARs that do not couple to Gs but do internalize (C-I+), and mutated BARs that do not couple to Gs and do not internalize (C-I-). For C+I+ and C-I+ cells, most of the receptors exhibited apparent affinities for the agonist isoproterenol that were 500-900-fold lower in equilibrium assays with intact cells than in short-time assays with intact cells or in equilibrium assays with isolated membranes, similar to previous results with cells expressing native BARs. The extent of conversion to this lower affinity form for C-I- cells was markedly decreased. Binding properties for the antagonist metoprolol were similar for all three BARs in both short-time and equilibrium assays. Isoproterenol competition in short-time and equilibrium assays also was compared in Chinese hamster fibroblasts expressing wild-type BARs, mutated BARs that lack BAR kinase sites, mutated BARs that lack cAMP-dependent protein kinase sites, and mutated BARs that lack both types of phosphorylation sites. All three BAR phosphorylation mutants showed only small but significant decreases, relative to the wild-type BAR, in the extent of conversion to the low affinity form. These results provide additional evidence that receptor internalization is the major determinant for the conversion of intact cell BARs to the low affinity form. Receptor phosphorylation may play a minor role in conversion to the low affinity form, whereas receptor coupling to Gs is apparently not required.
在对完整细胞进行平衡竞争结合试验的过程中，激动剂可诱导β - 肾上腺素能受体（BARs）从对激动剂具有高亲和力的天然形式转变为表观亲和力明显较低的形式。研究了受体内化、受体与Gs偶联以及受体磷酸化在这种激动剂诱导的向低亲和力形式转变中的作用。在表达野生型BARs（C + I +）、不与Gs偶联但可内化的突变型BARs（C - I +）以及不与Gs偶联且不内化的突变型BARs（C - I -）的小鼠L细胞中，测定了激动剂和拮抗剂对[¹²⁵I]碘吲哚洛尔与完整细胞结合的竞争情况。对于C + I +和C - I +细胞，在完整细胞的平衡试验中，大多数受体对激动剂异丙肾上腺素的表观亲和力比在完整细胞的短时间试验或分离膜的平衡试验中低500 - 900倍，这与之前表达天然BARs的细胞的结果相似。C - I -细胞向这种较低亲和力形式的转变程度明显降低。在短时间和平衡试验中，所有三种BARs对拮抗剂美托洛尔的结合特性相似。在表达野生型BARs、缺乏BAR激酶位点的突变型BARs、缺乏cAMP依赖性蛋白激酶位点的突变型BARs以及缺乏两种磷酸化位点的突变型BARs的中国仓鼠成纤维细胞中，也比较了短时间和平衡试验中的异丙肾上腺素竞争情况。相对于野生型BAR，所有三种BAR磷酸化突变体向低亲和力形式的转变程度仅出现了小幅度但显著的降低。这些结果提供了额外的证据，表明受体内化是完整细胞BARs转变为低亲和力形式的主要决定因素。受体磷酸化在向低亲和力形式的转变中可能起次要作用，而受体与Gs的偶联显然不是必需的。
